Identification of the Lyso-Form N-Acyl Intramolecular Transferase in Low-GC Firmicutes

ABSTRACT Bacterial lipoproteins are embedded in the cell membrane of both Gram-positive and Gram-negative bacteria, where they serve numerous functions central to cell envelope physiology. Lipoproteins are tethered to the membrane by an N-acyl-S-(mono/di)-acyl-glyceryl-cysteine anchor that is variously acylated depending on the genus. In several low-GC, Gram-positive firmicutes, a monoacyl-glyceryl-cysteine with an N-terminal fatty acid (known as the lyso form) has been reported, though how it is formed is unknown. Here, through an intergenic complementation rescue assay in Escherichia coli, we report the identification of a common orthologous transmembrane protein in both Enterococcus faecalis and Bacillus cereus that is capable of forming lyso-form lipoproteins. When deleted from the native host, lipoproteins remain diacylated with a free N terminus, as maturation to the N-acylated lyso form is abolished. Evidence is presented suggesting that the previously unknown gene product functions through a novel intramolecular transacylation mechanism, transferring a fatty acid from the diacylglycerol moiety to the α-amino group of the lipidated cysteine. As such, the discovered gene has been named lipoprotein intramolecular transacylase (lit), to differentiate it from the gene for the intermolecular N-acyltransferase (lnt) involved in triacyl lipoprotein biosynthesis in Gram-negative organisms. IMPORTANCE This study identifies a new enzyme, conserved among low-GC, Gram-positive bacteria, that is involved in bacterial lipoprotein biosynthesis and synthesizes lyso-form lipoproteins. Its discovery is an essential first step in determining the physiological role of N-terminal lipoprotein acylation in Gram-positive bacteria and how these modifications impact bacterial cell envelope function.

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Linearized esculentin-2EM shows pH dependent antibacterial activity with an alkaline optimum

Molecular and Cellular Biochemistry ◽

10.1007/s11010-021-04181-7 ◽

2021 ◽

Author(s):

Erum Malik ◽

David A. Phoenix ◽

Timothy J. Snape ◽

Frederick Harris ◽

Jaipaul Singh ◽

...

Keyword(s):

Helical Structure ◽

Food Preservation ◽

Forming Process ◽

Gram Positive ◽

Gram Negative ◽

Bacterial Membranes ◽

Gram Positive Bacteria ◽

Alkaline Conditions ◽

Ph Dependent ◽

Gram Negative Organisms

AbstractHere the hypothesis that linearized esculentin 2EM (E2EM-lin) from Glandirana emeljanovi possesses pH dependent activity is investigated. The peptide showed weak activity against Gram-negative bacteria (MLCs ≥ 75.0 μM) but potent efficacy towards Gram-positive bacteria (MLCs ≤ 6.25 μM). E2EM-lin adopted an α-helical structure in the presence of bacterial membranes that increased as pH was increased from 6 to 8 (↑ 15.5–26.9%), whilst similar increases in pH enhanced the ability of the peptide to penetrate (↑ 2.3–5.1 mN m−1) and lyse (↑ 15.1–32.5%) these membranes. Theoretical analysis predicted that this membranolytic mechanism involved a tilted segment, that increased along the α-helical long axis of E2EM-lin (1–23) in the N → C direction, with − < µH > increasing overall from circa − 0.8 to − 0.3. In combination, these data showed that E2EM-lin killed bacteria via novel mechanisms that were enhanced by alkaline conditions and involved the formation of tilted and membranolytic, α-helical structure. The preference of E2EM-lin for Gram-positive bacteria over Gram-negative organisms was primarily driven by the superior ability of phosphatidylglycerol to induce α-helical structure in the peptide as compared to phosphatidylethanolamine. These data were used to generate a novel pore-forming model for the membranolytic activity of E2EM-lin, which would appear to be the first, major reported instance of pH dependent AMPs with alkaline optima using tilted structure to drive a pore-forming process. It is proposed that E2EM-lin has the potential for development to serve purposes ranging from therapeutic usage, such as chronic wound disinfection, to food preservation by killing food spoilage organisms.

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Fusobacterium nucleatum, the first Gram-negative bacterium demonstrated to produce polyglutamate

Canadian Journal of Microbiology ◽

10.1139/w09-003 ◽

2009 ◽

Vol 55 (5) ◽

pp. 627-632 ◽

Cited By ~ 33

Author(s):

Thomas Candela ◽

Marie Moya ◽

Michel Haustant ◽

Agnès Fouet

Keyword(s):

In Silico Analysis ◽

Fusobacterium Nucleatum ◽

Gram Negative Bacteria ◽

Negative Bacterium ◽

Gram Positive ◽

Gram Negative ◽

Gram Positive Bacteria ◽

The Third ◽

Gram Negative Organisms ◽

First Time

Poly-γ-glutamate has been described in many Gram-positive organisms. When anchored to the surface, it is a capsule and as such a virulence factor. Based on sequence similarities, few Gram-negative organisms have been suggested to synthesize poly-γ-glutamate. For the first time, a Gram-negative bacterium, Fusobacterium nucleatum , is shown to produce and secrete poly-γ-glutamate. Putative poly-γ-glutamate-synthesizing genes from Gram-negative organisms have been compared with their Gram-positive homologs by in silico analysis, i.e., gene sequence and phylogenetic analysis. Clusters of three instead of four genes were highlighted by our screen. The products of the first two genes display similarity with their Gram-positive equivalents, yet the sequences from the Gram-negative organisms can be distinguished from those of the Gram-positives. Interestingly, the sequence of the predicted product of the third gene is conserved among Gram-negative bacteria but displays no similarity to that of either the third or fourth gene of the Gram-positive operons. It is suggested that, like for Gram-positive bacteria, poly-γ-glutamate has a role in virulence for pathogens and one in survival for other Gram-negative bacteria.

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The Outer Surface Lipoprotein VolA Mediates Utilization of Exogenous Lipids by Vibrio cholerae

mBio ◽

10.1128/mbio.00305-13 ◽

2013 ◽

Vol 4 (3) ◽

Cited By ~ 23

Author(s):

Aaron C. Pride ◽

Carmen M. Herrera ◽

Ziqiang Guan ◽

David K. Giles ◽

M. Stephen Trent

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Carbon Source ◽

Outer Membrane ◽

Vibrio Cholerae ◽

Homeoviscous Adaptation ◽

Gram Negative ◽

Content Type ◽

Enzymatic Function ◽

Gram Negative Organisms

ABSTRACTPrevious work from our laboratory showed that the Gram-negative aquatic pathogenVibrio choleraecan take up a much wider repertoire of fatty acids than other Gram-negative organisms. The current work elaborated on the ability ofV. choleraeto exploit an even more diverse pool of lipid nutrients from its environment. We have demonstrated that the bacterium can use lysophosphatidylcholine as a metabolite for growth. Using a combination of thin-layer chromatography and mass spectrometry, we also showed that lysophosphatidylcholine-derived fatty acid moieties can be used for remodeling theV. choleraemembrane architecture. Furthermore, we have identified a lysophospholipase, VolA (Vibrioouter membrane lysophospholipase A), required for these activities. The enzyme is well conserved inVibriospecies, is coexpressed with the outer membrane fatty acid transporter FadL, is one of very few surface-exposed lipoprotein enzymes to be identified in Gram-negative bacteria and the first instance of a surface lipoprotein phospholipase. We propose a model whereby the bacterium efficiently couples the liberation of fatty acid from lysophosphatidylcholine to its subsequent metabolic uptake. An expanded ability to scavenge diverse environmental lipids at the bacterial surface increases overall bacterial fitness and promotes homeoviscous adaptation through membrane remodeling.IMPORTANCEOur understanding of how bacteria utilize environmental lipid sources has been limited to lipids such as fatty acids and cholesterol. This narrow scope may be attributed to both the intricate nature of lipid uptake mechanisms and the diversity of lipid substrates encountered within an ecological niche. By examining the ability of the pathogenVibrio choleraeto utilize exogenous lipids, we uncovered a surface-exposed lipoprotein (VolA) that is required for processing the prevalent host lipid lysophosphatidylcholine. VolA functions as a lipase liberating a fatty acid from exogenous lysophospholipids. The freed fatty acid is then transported into the cell, serving as a carbon source, or shunted into phospholipid synthesis for membrane assembly. A limited number of surface-exposed lipoproteins have been found in Gram-negative organisms, and few have enzymatic function. This work highlights the ability of bacteria to exploit exogenous lipids for both maintenance of the membrane and carbon source acquisition.

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Recognition of Streptococcus pneumoniae and Muramyl Dipeptide by NOD2 Results in Potent Induction of MMP-9, Which Can Be Controlled by Lipopolysaccharide Stimulation

Infection and Immunity ◽

10.1128/iai.02150-14 ◽

2014 ◽

Vol 82 (12) ◽

pp. 4952-4958 ◽

Cited By ~ 10

Author(s):

Marloes Vissers ◽

Yvonne Hartman ◽

Laszlo Groh ◽

Dirk J. de Jong ◽

Marien I. de Jonge ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Mononuclear Cells ◽

Muramyl Dipeptide ◽

Tissue Inhibitor Of Metalloproteinase ◽

Gram Negative Bacteria ◽

Gram Positive ◽

Potent Inducer ◽

Gram Negative ◽

Content Type ◽

Gram Positive Bacteria

ABSTRACTMatrix metallopeptidase 9 (MMP-9) is a protease involved in the degradation of extracellular matrix collagen. Evidence suggests that MMP-9 is involved in pathogenesis duringStreptococcus pneumoniaeinfection. However, not much is known about the induction of MMP-9 and the regulatory processes involved. We show here that the Gram-positive bacteria used in this study induced large amounts of MMP-9, in contrast to the Gram-negative bacteria that were used. An important pathogen-associated molecular pattern (PAMP) for Gram-positive bacteria is muramyl dipeptide (MDP). MDP is a very potent inducer of MMP-9 and showed a dose-dependent MMP-9 induction. Experiments using peripheral blood mononuclear cells (PBMCs) from Crohn's disease patients with nonfunctional NOD2 showed that MMP-9 induction byStreptococcus pneumoniaeand MDP is NOD2 dependent. Increasing amounts of lipopolysaccharide (LPS), an important PAMP for Gram-negative bacteria, resulted in decreasing amounts of MMP-9. Moreover, the induction of MMP-9 by MDP could be counteracted by simultaneously adding LPS. The inhibition of MMP-9 expression by LPS was found to be regulated posttranscriptionally, independently of tissue inhibitor of metalloproteinase 1 (TIMP-1), an endogenous inhibitor of MMP-9. Collectively, these data show thatStreptococcus pneumoniaeis able to induce large amounts of MMP-9. These high MMP-9 levels are potentially involved inStreptococcus pneumoniaepathogenesis.

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Distinctive Binding of Avibactam to Penicillin-Binding Proteins of Gram-Negative and Gram-Positive Bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02102-15 ◽

2015 ◽

Vol 60 (2) ◽

pp. 752-756 ◽

Cited By ~ 34

Author(s):

Abdelhamid Asli ◽

Eric Brouillette ◽

Kevin M. Krause ◽

Wright W. Nichols ◽

François Malouin

Keyword(s):

Binding Proteins ◽

New Combination ◽

Gram Negative Bacteria ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

Penicillin Binding Proteins ◽

Gram Positive Bacteria ◽

Fixed Concentration ◽

Competition Assays

ABSTRACTAvibactam is a novel non-β-lactam β-lactamase inhibitor that covalently acylates a variety of β-lactamases, causing inhibition. Although avibactam presents limited antibacterial activity, its acylation ability toward bacterial penicillin-binding proteins (PBPs) was investigated.Staphylococcus aureuswas of particular interest due to the reported β-lactamase activity of PBP4. The binding of avibactam to PBPs was measured by adding increasing concentrations to membrane preparations of a variety of Gram-positive and Gram-negative bacteria prior to addition of the fluorescent reagent Bocillin FL. Relative binding (measured here as the 50% inhibitory concentration [IC50]) to PBPs was estimated by quantification of fluorescence after gel electrophoresis. Avibactam was found to selectively bind to some PBPs. InEscherichia coli,Pseudomonas aeruginosa,Haemophilus influenzae, andS. aureus, avibactam primarily bound to PBP2, with IC50s of 0.92, 1.1, 3.0, and 51 μg/ml, respectively, whereas binding to PBP3 was observed inStreptococcus pneumoniae(IC50, 8.1 μg/ml). Interestingly, avibactam was able to significantly enhance labeling ofS. aureusPBP4 by Bocillin FL. In PBP competition assays withS. aureus, where avibactam was used at a fixed concentration in combination with varied amounts of ceftazidime, the apparent IC50of ceftazidime was found to be very similar to that determined for ceftazidime when used alone. In conclusion, avibactam is able to covalently bind to some bacterial PBPs. Identification of those PBP targets may allow the development of new diazabicyclooctane derivatives with improved affinity for PBPs or new combination therapies that act on multiple PBP targets.

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Secretory Phospholipase A2 Is the Principal Bactericide for Staphylococci and Other Gram-Positive Bacteria in Human Tears

Infection and Immunity ◽

10.1128/iai.66.6.2791-2797.1998 ◽

1998 ◽

Vol 66 (6) ◽

pp. 2791-2797 ◽

Cited By ~ 172

Author(s):

Xiao-Dan Qu ◽

Robert I. Lehrer

Keyword(s):

Bactericidal Activity ◽

Tear Film ◽

Phospholipase A ◽

Secretory Phospholipase A2 ◽

Gram Positive ◽

Gram Negative ◽

Secretory Phospholipase ◽

Calcium Dependent ◽

Gram Positive Bacteria ◽

Gram Negative Organisms

ABSTRACT We examined human tears for molecules that killed gram-positive bacteria. The principal mediator of bactericidal activity against staphylococci proved to be a calcium-dependent enzyme, secretory phospholipase A2. Whereas the concentration of secretory phospholipase A2 in the normal tear film exceeded 30 μg/ml, only 1.1 ng (<0.1 nM) of the enzyme per ml sufficed to killListeria monocytogenes and 15 to 80 ng/ml killedStaphylococcus aureus. Despite its efficacy against gram-positive bacteria, secretory phospholipase A2 lacked bactericidal activity against gram-negative organisms (Escherichia coli, Salmonella typhimurium, andPseudomonas aeruginosa) when tested in the ionic environment of tears. Given the presence of secretory phospholipase A2 in tears, intestinal secretions, and leukocytes, this enzyme may play a substantial role in innate mucosal and systemic bactericidal defenses against gram-positive bacteria.

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Effect of Lactose Monolaurate on Pathogenic and Nonpathogenic Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.07701-11 ◽

2012 ◽

Vol 78 (9) ◽

pp. 3465-3468 ◽

Cited By ~ 22

Author(s):

Ashwini Wagh ◽

Shujie Shen ◽

Fen Ann Shen ◽

Charles D. Miller ◽

Marie K. Walsh

Keyword(s):

Listeria Monocytogenes ◽

Antimicrobial Activities ◽

Gram Negative Bacteria ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

Gram Positive Bacteria ◽

Lactose Monolaurate

ABSTRACTThe antimicrobial activities of sucrose monolaurate and a novel ester, lactose monolaurate (LML), were tested. Gram-positive bacteria were more susceptible than Gram-negative bacteria to both esters. The minimal bactericidal concentrations of LML were 5 to 9.5 mM forListeria monocytogenesisolates and 0.2 to 2 mM forMycobacteriumisolates.

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Induction of Daptomycin Tolerance in Enterococcus faecalis by Fatty Acid Combinations

Applied and Environmental Microbiology ◽

10.1128/aem.01178-20 ◽

2020 ◽

Vol 86 (20) ◽

Author(s):

William Brewer ◽

Johnathan Harrison ◽

Holly E. Saito ◽

Elizabeth M. Fozo

Keyword(s):

Fatty Acids ◽

Antibiotic Resistance ◽

Fatty Acid ◽

Stearic Acid ◽

Enterococcus Faecalis ◽

Cell Envelope ◽

Negative Effects ◽

Gram Positive ◽

Bacterial Populations ◽

Content Type

ABSTRACT Enterococcus faecalis is a Gram-positive bacterium that normally exists as an intestinal commensal in humans but is also a leading cause of nosocomial infections. Previous work noted that growth supplementation with serum induced tolerance to membrane-damaging agents, including the antibiotic daptomycin. Specific fatty acids found within serum could independently provide tolerance to daptomycin (protective fatty acids), yet some fatty acids found in serum did not and had negative effects on enterococcal physiology (nonprotective fatty acids). Here, we measured a wide array of physiological responses after supplementation with combinations of protective and nonprotective fatty acids to better understand how serum induces daptomycin tolerance. When cells were supplemented with either nonprotective fatty acid, palmitic acid, or stearic acid, there were marked defects in growth and morphology, but these defects were rescued upon supplementation with either protective fatty acid, oleic acid, or linoleic acid. Membrane fluidity decreased with growth in either palmitic or stearic acid alone but returned to basal levels when a protective fatty acid was supplied. Daptomycin tolerance could be induced if a protective fatty acid was provided with a nonprotective fatty acid, and some specific combinations protected as well as serum supplementation. While cell envelope charge has been associated with tolerance to daptomycin in other Gram-positive bacteria, we concluded that it does not correlate with the fatty acid-induced protection we observed. Based on these observations, we conclude that daptomycin tolerance by serum is driven by specific, protective fatty acids found within the fluid. IMPORTANCE With an increasing prevalence of antibiotic resistance in the clinic, we strive to understand more about microbial defensive mechanisms. A nongenetic tolerance to the antibiotic daptomycin was discovered in Enterococcus faecalis that results in the increased survival of bacterial populations after treatment with the drug. This tolerance mechanism likely synergizes with antibiotic resistance in the clinic. Given that this tolerance phenotype is induced by incorporation of fatty acids present in the host, it can be assumed that infections by this organism require a higher dose of antibiotic for successful eradication. The mixture of fatty acids in human fluids is quite diverse, with little understanding between the interplay of fatty acid combinations and the tolerance phenotype we observe. It is crucial to understand the effects of fatty acid combinations on E. faecalis physiology if we are to suppress the tolerance physiology in the clinic.

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Development of Methionyl-tRNA Synthetase Inhibitors as Antibiotics for Gram-Positive Bacterial Infections

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00999-17 ◽

2017 ◽

Vol 61 (11) ◽

Cited By ~ 8

Author(s):

Omeed Faghih ◽

Zhongsheng Zhang ◽

Ranae M. Ranade ◽

J. Robert Gillespie ◽

Sharon A. Creason ◽

...

Keyword(s):

Protein Synthesis ◽

Bacterial Species ◽

Bacterial Load ◽

Trna Synthetase ◽

Resistant Bacteria ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

Gram Positive Bacteria ◽

Methionyl Trna Synthetase

ABSTRACT Antibiotic-resistant bacteria are widespread and pose a growing threat to human health. New antibiotics acting by novel mechanisms of action are needed to address this challenge. The bacterial methionyl-tRNA synthetase (MetRS) enzyme is essential for protein synthesis, and the type found in Gram-positive bacteria is substantially different from its counterpart found in the mammalian cytoplasm. Both previously published and new selective inhibitors were shown to be highly active against Gram-positive bacteria with MICs of ≤1.3 μg/ml against Staphylococcus, Enterococcus, and Streptococcus strains. Incorporation of radioactive precursors demonstrated that the mechanism of activity was due to the inhibition of protein synthesis. Little activity against Gram-negative bacteria was observed, consistent with the fact that Gram-negative bacterial species contain a different type of MetRS enzyme. The ratio of the MIC to the minimum bactericidal concentration (MBC) was consistent with a bacteriostatic mechanism. The level of protein binding of the compounds was high (>95%), and this translated to a substantial increase in MICs when the compounds were tested in the presence of serum. Despite this, the compounds were very active when they were tested in a Staphylococcus aureus murine thigh infection model. Compounds 1717 and 2144, given by oral gavage, resulted in 3- to 4-log decreases in the bacterial load compared to that in vehicle-treated mice, which was comparable to the results observed with the comparator drugs, vancomycin and linezolid. In summary, the research describes MetRS inhibitors with oral bioavailability that represent a class of compounds acting by a novel mechanism with excellent potential for clinical development.

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Chitosan Augments Photodynamic Inactivation of Gram-Positive and Gram-Negative Bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00550-10 ◽

2011 ◽

Vol 55 (5) ◽

pp. 1883-1890 ◽

Cited By ~ 41

Author(s):

Tsuimin Tsai ◽

Hsiung-Fei Chien ◽

Tze-Hsien Wang ◽

Ching-Tsan Huang ◽

Yaw-Bee Ker ◽

...

Keyword(s):

Staphylococcus Epidermidis ◽

Light Exposure ◽

Gram Negative Bacteria ◽

Photodynamic Inactivation ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

Gram Positive Bacteria ◽

Microbial Infections ◽

Promising Treatment

ABSTRACTAntimicrobial photodynamic inactivation (PDI) was shown to be a promising treatment modality for microbial infections. This study explores the effect of chitosan, a polycationic biopolymer, in increasing the PDI efficacy against Gram-positive bacteria, includingStaphylococcus aureus,Staphylococcus epidermidis,Streptococcus pyogenes, and methicillin-resistantS. aureus(MRSA), as well as the Gram-negative bacteriaPseudomonas aeruginosaandAcinetobacter baumannii. Chitosan at <0.1% was included in the antibacterial process either by coincubation with hematoporphyrin (Hp) and subjection to light exposure to induce the PDI effect or by addition after PDI and further incubation for 30 min. Under conditions in which Hp-PDI killed the microbe on a 2- to 4-log scale, treatment with chitosan at concentrations of as low as 0.025% for a further 30 min completely eradicated the bacteria (which were originally at ∼108CFU/ml). Similar results were also found with toluidine blue O (TBO)-mediated PDI in planktonic and biofilm cells. However, without PDI treatment, chitosan alone did not exert significant antimicrobial activity with 30 min of incubation, suggesting that the potentiated effect of chitosan worked after the bacterial damage induced by PDI. Further studies indicated that the potentiated PDI effect of chitosan was related to the level of PDI damage and the deacetylation level of the chitosan. These results indicate that the combination of PDI and chitosan is quite promising for eradicating microbial infections.

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