Protein Interactomes Identify Distinct Pathways for Streptococcus mutans YidC1 and YidC2 Membrane Protein Insertases

AbstractVirulence properties of cariogenic Streptococcus mutans depend on integral membrane proteins. Bacterial protein trafficking involves the co-translational signal recognition particle (SRP) pathway components Ffh and FtsY, the SecY translocon, and membrane-localized YidC chaperone/insertases. Unlike Escherichia coli, S. mutans survives loss of the SRP pathway. In addition, S. mutans has two yidC paralogs. The ΔyidC2 phenotype largely parallels that of Δffh and ΔftsY while the ΔyidC1 phenotype is less severe. This study defined YidC1 and YidC2 interactomes to identify their respective functions alone and in concert with the SRP, ribosome, and/or Sec translocon. A chemical cross-linking approach was employed, whereby whole cell lysates were treated with formaldehyde followed by Western blotting using anti-Ffh, FtsY, YidC1 or YidC2 antibodies and mass spectrometry (MS) analysis of gel-shifted bands. Cross-linked lysates from WT and ΔyidC2 strains were also reacted with anti-YidC2 antibodies coupled to magnetic Dynabeads™, with co-captured proteins identified by MS. Additionally, C-terminal tails of YidC1 and YidC2 were engineered as glutathione-S-transferase fusion proteins and subjected to 2D Difference Gel Electrophoresis and MS analysis after being reacted with non-cross-linked lysates. Results indicate that YidC2 works in concert with the SRP-pathway, while YidC1 works in concert with the SecY translocon independently of the SRP. In addition, YidC1 and/or YidC2 can act alone in the insertion of a limited number of small integral membrane proteins. The YidC2-SRP and YidC1/SecY pathways appear to function as part of an integrated machinery that couples translation and transport with cell division, as well as transcription and DNA replication.ImportanceStreptococcus mutans is a prevalent oral pathogen and causative agent of tooth decay. Many proteins that enable this bacterium to thrive in its environmental niche, and cause disease, are embedded in its cytoplasmic membrane. The machinery that transports proteins into bacterial membranes differs between Gram-negative and Gram-positive organisms. One important difference is the presence of multiple YidC paralogs in Gram-positive bacteria. Characterization of a protein’s interactome can help define its physiological role. Herein, we characterized the interactomes of S. mutans YidC1 and YidC2. Results indicate that YidC1 and YidC2 have individualized functions in separate membrane insertion pathways, and suggest putative substrates of the respective pathways. Furthermore, S. mutans membrane transport proteins appear as part of a larger network of proteins involved in replication, transcription, translation, and cell division/cell shape. This information contributes to our understanding of protein transport in Gram-positive bacteria in general, and informs our understanding of S. mutans pathogenesis.

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Protein Interactomes of Streptococcus mutans YidC1 and YidC2 Membrane Protein Insertases Suggest SRP Pathway-Independent- and -Dependent Functions, Respectively

mSphere ◽

10.1128/msphere.01308-20 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

Patricia Lara Vasquez ◽

Surabhi Mishra ◽

Senthil K. Kuppuswamy ◽

Paula J. Crowley ◽

L. Jeannine Brady

Keyword(s):

Streptococcus Mutans ◽

Large Network ◽

Gram Positive ◽

Western Blots ◽

Content Type ◽

Gram Positive Bacteria ◽

Binding Partners ◽

Ms Analysis ◽

Sec Translocon ◽

Two Hybrid

ABSTRACT Virulence properties of cariogenic Streptococcus mutans depend on integral membrane proteins. Bacterial cotranslational protein trafficking involves the signal recognition particle (SRP) pathway components Ffh and FtsY, the SecYEG translocon, and YidC chaperone/insertases. Unlike Escherichia coli, S. mutans survives loss of the SRP pathway and has two yidC paralogs. This study characterized YidC1 and YidC2 interactomes to clarify respective functions alone and in concert with the SRP and/or Sec translocon. Western blots of formaldehyde cross-linked or untreated S. mutans lysates were reacted with anti-Ffh, anti-FtsY, anti-YidC1, or anti-YidC2 antibodies followed by mass spectrometry (MS) analysis of gel-shifted bands. Cross-linked lysates of wild-type and ΔyidC2 strains were reacted with anti-YidC2-coupled Dynabeads, and cocaptured proteins were identified by MS. Last, YidC1 and YidC2 C-terminal tail-captured proteins were subjected to two-dimensional (2D) difference gel electrophoresis and MS analysis. Direct interactions of putative YidC1 and YidC2 binding partners were confirmed by bacterial two-hybrid assay. Our results suggest YidC2 works preferentially with the SRP pathway, while YidC1 is preferred for SRP-independent Sec translocon-mediated translocation. YidC1 and YidC2 autonomous pathways were also apparent. Two-hybrid assay identified interactions between holotranslocon components SecYEG/YajC and YidC1. Both YidC1 and YidC2 interacted with Ffh, FtsY, and chaperones DnaK and RopA. Putative membrane-localized substrates HlyX, LemA, and SMU_591c interacted with both YidC1 and YidC2. Identification of several Rgp proteins in the YidC1 interactome suggested its involvement in bacitracin resistance, which was decreased in ΔyidC1 and SRP-deficient mutants. Collectively, YidC1 and YidC2 interactome analyses has further distinguished these paralogs in the Gram-positive bacterium S. mutans. IMPORTANCE Streptococcus mutans is a prevalent oral pathogen and major causative agent of tooth decay. Many proteins that enable this bacterium to thrive in its environmental niche and cause disease are embedded in its cytoplasmic membrane. The machinery that transports proteins into bacterial membranes differs between Gram-negative and Gram-positive organisms, an important difference being the presence of multiple YidC paralogs in Gram-positive bacteria. Characterization of a protein’s interactome can help define its physiological role. Herein, we characterized the interactomes of S. mutans YidC1 and YidC2. Results demonstrated substantial overlap between their interactomes but also revealed several differences in their direct protein binding partners. Membrane transport machinery components were identified in the context of a large network of proteins involved in replication, transcription, translation, and cell division/cell shape. This information contributes to our understanding of protein transport in Gram-positive bacteria in general and informs our understanding of S. mutans pathogenesis.

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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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How Teichoic Acids Could Support a Periplasm in Gram-Positive Bacteria, and Let Cell Division Cheat Turgor Pressure

Frontiers in Microbiology ◽

10.3389/fmicb.2021.664704 ◽

2021 ◽

Vol 12 ◽

Author(s):

Harold P. Erickson

Keyword(s):

Cell Division ◽

Charge Density ◽

Turgor Pressure ◽

Support Pressure ◽

Periplasmic Space ◽

Gram Positive ◽

Teichoic Acids ◽

Gram Positive Bacteria ◽

Peptidoglycan Cell Wall ◽

Anionic Charge

The cytoplasm of bacteria is maintained at a higher osmolality than the growth medium, which generates a turgor pressure. The cell membrane (CM) cannot support a large turgor, so there are two possibilities for transferring the pressure to the peptidoglycan cell wall (PGW): (1) the CM could be pressed directly against the PGW, or (2) the CM could be separated from the PGW by a periplasmic space that is isoosmotic with the cytoplasm. There is strong evidence for gram-negative bacteria that a periplasm exists and is isoosmotic with the cytoplasm. No comparable studies have been done for gram-positive bacteria. Here I suggest that a periplasmic space is probably essential in order for the periplasmic proteins to function, including especially the PBPs that remodel the peptidoglycan wall. I then present a semi-quantitative analysis of how teichoic acids could support a periplasm that is isoosmotic with the cytoplasm. The fixed anionic charge density of teichoic acids in the periplasm is ∼0.5 M, which would bring in ∼0.5 M Na+ neutralizing ions. This approximately balances the excess osmolality of the cytoplasm that would produce a turgor pressure of 19 atm. The 0.5 M fixed charge density is similar to that of proteoglycans in articular cartilage, suggesting a comparability ability to support pressure. An isoosmotic periplasm would be especially important for cell division, since it would allow CM constriction and PGW synthesis to avoid turgor pressure.

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Effects of Antibacterial Peptide F1 on Bacterial Liposome Membrane Integrity

Frontiers in Nutrition ◽

10.3389/fnut.2021.768890 ◽

2021 ◽

Vol 8 ◽

Author(s):

Qun Wang ◽

Bo Peng ◽

Mingyue Song ◽

Abdullah ◽

Jun Li ◽

...

Keyword(s):

Antimicrobial Peptide ◽

Membrane Structure ◽

Bacterial Membrane ◽

Phospholipid Membranes ◽

Phospholipid Membrane ◽

Gram Positive ◽

Liposome Membrane ◽

Gram Negative ◽

Bacterial Membranes ◽

Gram Positive Bacteria

Previous studies from our lab have shown that the antimicrobial peptide F1 obtained from the milk fermentation by Lactobacillus paracasei FX-6 derived from Tibetan kefir was different from common antimicrobial peptides; specifically, F1 simultaneously inhibited the growth of Gram-negative and Gram-positive bacteria. Here, we present follow-on work demonstrating that after the antimicrobial peptide F1 acts on either Escherichia coli ATCC 25922 (E. coli) or Staphylococcus aureus ATCC 63589 (S. aureus), their respective bacterial membranes were severely deformed. This deformation allowed leakage of potassium and magnesium ions from the bacterial membrane. The interaction between the antimicrobial peptide F1 and the bacterial membrane was further explored by artificially simulating the bacterial phospholipid membranes and then extracting them. The study results indicated that after the antimicrobial peptide F1 interacted with the bacterial membranes caused significant calcein leakage that had been simulated by different liposomes. Furthermore, transmission electron microscopy observations revealed that the phospholipid membrane structure was destroyed and the liposomes presented aggregation and precipitation. Quartz Crystal Microbalance with Dissipation (QCM-D) results showed that the antimicrobial peptide F1 significantly reduced the quality of liposome membrane and increased their viscoelasticity. Based on the study's findings, the phospholipid membrane particle size was significantly increased, indicating that the antimicrobial peptide F1 had a direct effect on the phospholipid membrane. Conclusively, the antimicrobial peptide F1 destroyed the membrane structure of both Gram-negative and Gram-positive bacteria by destroying the shared components of their respective phospholipid membranes which resulted in leakage of cell contents and subsequently cell death.

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Second Harmonic Generation Spectroscopy of Membrane Probe Dynamics in Gram-Positive Bacteria

10.1101/645788 ◽

2019 ◽

Author(s):

L. N. Miller ◽

W. T. Brewer ◽

J. D. Williams ◽

E. M. Fozo ◽

T. R. Calhoun

Keyword(s):

Second Harmonic Generation ◽

Small Molecules ◽

Harmonic Generation ◽

Second Harmonic ◽

Bacterial Species ◽

Gram Positive ◽

Flip Flop ◽

Bacterial Membranes ◽

Gram Positive Bacteria ◽

Fluorescence Measurements

ABSTRACTBacterial membranes are complex mixtures with dispersity that is dynamic over scales of both space and time. In order to capture adsorption onto and transport within these mixtures, we conduct simultaneous second harmonic generation (SHG) and two photon fluorescence measurements on two different gram-positive bacterial species as the cells uptake membrane-specific probe molecules. Our results show that SHG can not only monitor the movement of small molecules across membrane leaflets, but is also sensitive to higher-level ordering of the molecules within the membrane. Further, we show that the membranes of Staphylococcus aureus remain more dynamic after longer times at room temperature in comparison to Enterococcus faecalis. Our findings provide insight into the variability of activities seen between structurally similar molecules in gram-positive bacteria while also demonstrating the power of SHG to examine these dynamics.STATEMENT OF SIGNIFICANCEBacterial membranes are highly adept at discerning and modifying their interactions with different small molecules in their environment. Here we show how second harmonic generation (SHG) spectroscopy can track the dynamics of structurally similar membrane probes in two gram-positive bacterial species. Our results reveal behavior that is dependent on both the probe molecule and the membrane composition. Specifically, we observe flip-flop between leaflets for one molecule, while the other molecule produces a signal indicative of larger scale ordering in the membrane. These phenomena can all be explained by considering potential differences in the membrane fluidity and surface charge between the two bacterial species. Overall, our work highlights the dynamic differences between bacterial membranes and SHG’s sensitivity to probing these systems.

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Alanyl-Phosphatidylglycerol and Lysyl-Phosphatidylglycerol Are Translocated by the Same MprF Flippases and Have Similar Capacities To Protect against the Antibiotic Daptomycin in Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00370-12 ◽

2012 ◽

Vol 56 (7) ◽

pp. 3492-3497 ◽

Cited By ~ 39

Author(s):

Christoph J. Slavetinsky ◽

Andreas Peschel ◽

Christoph M. Ernst

Keyword(s):

Staphylococcus Aureus ◽

Deletion Mutant ◽

Wild Type ◽

Gram Positive ◽

Content Type ◽

Wild Type Level ◽

Bacterial Membranes ◽

Gram Positive Bacteria ◽

Cytoplasmic Membranes ◽

Simultaneous Expression

ABSTRACTThe lysinylation of negatively charged phosphatidylglycerol by MprF proteins reduces the affinity of cationic antimicrobial peptides (CAMPs) for bacterial cytoplasmic membranes and reduces the susceptibility of several Gram-positive bacterial pathogens to CAMPs. MprF ofStaphylococcus aureusencompasses a lysyl-phosphatidylglycerol (Lys-PG) synthase and a Lys-PG flippase domain. In contrast,Clostridium perfringensencodes two MprF homologs which specifically synthesize alanyl-phosphatidylglycerol (Ala-PG) or Lys-PG, while only the Lys-PG synthase is fused to a putative flippase domain. It remains unknown whether cationic Lys-PG and zwitterionic Ala-PG differ in their capacities to be translocated by MprF flippases and if both can reduce CAMP susceptibility in Gram-positive bacteria. By expressing the MprF proteins ofC. perfringensin anS. aureus mprFdeletion mutant, we found that both lipids can be efficiently produced inS. aureus. Simultaneous expression of the Lys-PG and Ala-PG synthases led to the production of both lipids and slightly increased the overall amounts of aminoacyl phospholipids. Ala-PG production by the correspondingC. perfringensenzyme did not affect susceptibility to CAMPs such as nisin and gallidermin or to the CAMP-like antibiotic daptomycin. However, coexpression of the Ala-PG synthase with flippase domains of Lys-PG synthesizing MprF proteins led to a wild-type level of daptomycin susceptibility, indicating that Ala-PG can also protect bacterial membranes against daptomycin and suggesting that Lys-PG flippases can also translocate the related lipid Ala-PG. Thus, bacterial aminoacyl phospholipid flippases exhibit more relaxed substrate specificity and Ala-PG and Lys-PG are more similar in their capacities to modulate membrane functions than anticipated.

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