No abstract

Historically, the O1 El Tor and classical biotypes of Vibrio cholerae have been differentiated by their resistance to the antimicrobial peptide polymyxin B. However, the molecular mechanisms associated with this phenotypic distinction have remained a mystery for 50 y. Both Gram-negative and Gram-positive bacteria modify their cell wall components with amine-containing substituents to reduce the net negative charge of the bacterial surface, thereby promoting cationic antimicrobial peptide resistance. In the present study, we demonstrate that V. cholerae modify the lipid A anchor of LPS with glycine and diglycine residues. This previously uncharacterized lipid A modification confers polymyxin resistance in V. cholerae El Tor, requiring three V. cholerae proteins: Vc1577 (AlmG), Vc1578 (AlmF), and Vc1579 (AlmE). Interestingly, the protein machinery required for glycine addition is reminiscent of the Gram-positive system responsible for D-alanylation of teichoic acids. Such machinery was not thought to be used by Gram-negative organisms. V. cholerae O1 El Tor mutants lacking genes involved in transferring glycine to LPS showed a 100-fold increase in sensitivity to polymyxin B. This work reveals a unique lipid A modification and demonstrates a charge-based remodeling strategy shared between Gram-positive and Gram-negative organisms.outer membrane | cell envelope | endotoxin | ultraviolet photodissociation T he Gram-negative pathogen Vibrio cholerae is the causative agent responsible for ∼300,000 reported cases annually of the severe diarrheal disease cholera. Since 1961, resistance to polymyxin B, a cationic antimicrobial peptide (CAMP), has been used to clinically differentiate between the two V. cholerae O1 biotypes, El Tor and classical (1). Interestingly the O1 classical biotype, which is polymyxin-sensitive, caused the first six cholera pandemics; however, polymyxin-resistant O1 El Tor strains are responsible for the current, seventh pandemic. Despite its importance, the molecular mechanisms accounting for this phenotypic difference have remained unknown for nearly 50 y.During infection, the cells of the innate immune system secrete CAMPs, which are small, positively charged proteins. Much like polymyxin, these peptides bind to and disrupt the bacterial cell membrane, ultimately resulting in death of the invading bacterial cell. Although Gram-negative and Gram-positive bacteria possess different cell wall structures, both have evolved mechanisms to remodel their cell envelope in response to CAMPs. These mechanisms often involve a common theme: neutralizing the negative charge of major cell wall components.The major surface component of Gram-negative bacteria is LPS, which is composed of three distinct regions: lipid A, core oligosaccharide, and O-antigen polysaccharide (2). Lipid A is the bioactive portion of LPS, which activates the human innate immune system through the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 complex (3). The negatively charged lipid A domain is synthesized through a well-...

193 nm ultraviolet photodissociation (UVPD) was implemented to sequence singly and multiply charged peptide anions. Upon dissociation by this method, a-/x-type, followed by d and w side-chain loss ions, were the most prolific and abundant sequence ions, often yielding 100% sequence coverage. The dissociation behavior of singly and multiply charged anions was significantly different with higher charged precursors yielding more sequence ions; however, all charge states investigated (1- through 3-) produced rich diagnostic information. UVPD at 193 nm was also shown to successfully differentiate and pinpoint labile phosphorylation modifications. The sequence ions were produced with high abundances, requiring limited averaging for satisfactory spectral quality. The intact, charge-reduced radical products generated by UV photoexcitation were also subjected to collision induced dissociation (termed, activated – electron photodetachment dissociation (a-EPD)), but UVPD alone yielded more predictable and higher abundance sequence ions. With the use of a basic (pH ~11.5), piperidine-modified mobile phase, LC-MS/UVPD was implemented and resulted in the successful analysis of mitogen-activated pathway kinases (MAPKs) using ultrafast activation times (5 nanoseconds).

Ultraviolet photodissociation (UVPD) at 193 nm was implemented on a linear ion trap mass spectrometer for high-throughput proteomic workflows. Upon irradiation by a single 5 ns laser pulse, efficient photodissociation of tryptic peptides was achieved with production of a, b, c, x, y, and z sequence ions, in addition to immonium ions and v and w side-chain loss ions. The factors that influence the UVPD mass spectra and subsequent in silico database searching via SEQUEST were evaluated. Peptide sequence aromaticity and the precursor charge state were found to influence photodissociation efficiency more so than the number of amide chromophores, and the ion trap q-value and number of laser pulses significantly affected the number and abundances of diagnostic product ions (e.g., sequence and immonium ions). Also, photoionization background subtraction was shown to dramatically improve SEQUEST results, especially when peptide signals were low. A liquid chromatography -mass spectrometry (LC-MS) -UVPD strategy was implemented and yielded comparable or better results relative to LC-MS -collision induced dissociation (CID) for analysis of proteolyzed bovine serum albumin and lysed human HT-1080 cytosolic fibrosarcoma cells.

Electron capture dissociation (ECD) [1] -an experiment generally performed within the high magnetic field of a Fourier transform ion cyclotron resonance mass spectrometerresults from the mutual storage of thermal electrons with multiply protonated peptide cations. The technique is particularly useful, as it generates random backbone cleavage with little regard to the presence of posttranslational modifications (PTMs), amino acid composition, or peptide length. Electron transfer dissociation (ETD), [2] the ion-ion analogue of ECD, is conducted in radio-frequency (RF) quadrupole ion trap devices, in which radical anions serve as electron donors. Since it can be implemented on virtually any mass spectrometer with an RF ion transfer or storage device, ETD has become an increasingly widespread dissociation method.The capture of an electron can trigger a free-radicaldriven rearrangement that results in N À C a backbone cleavage and the production of c-and zC-type fragment ions. Sometimes, however, the precursor cation captures the electron and forms a long-lived, charge-reduced species that does not separate (an ECnoD or ETnoD product).[3] This phenomenon becomes more probable as the mass-to-charge (m/z) ratio of the precursor increases. As the charge density decreases, the magnitude of intramolecular noncovalent interactions increases, so that the newly formed c-and zC-type fragment ions often remain bound following electron capture and cleavage-an obstacle of higher consequence for ETD, which is conducted under conditions of elevated pressure.[4] McLafferty and co-workers reported that photon bombardment of the precursor cation prior to ECD (activated-ion ECD, AI ECD) decreased nondissociative electron capture, [5] presumably by destroying the secondary structure of the peptide cation prior to electron capture.ETD is conducted at pressures that are approximately 10 6 times higher than those used in ECD (which is carried out at approximately 0.13 Pa). Therefore, precursor cations undergoing ETD are considerably cooler, and preactivation either with photons or through collisions is expected to produce only short-lived (< 1 ms) unfolding. Recently, we examined the use of collisions to coerce the ETnoD products into dissociating through a technique coined ETcaD (ETD in conjunction with collisional activation).[3] The method increased the number and intensity of N À C a backbone cleavages; however, the majority of the newly formed fragment ions displayed evidence of hydrogen-atom rearrangement to produce evenelectron z-type fragments and odd-electron cC-type products. ECD practitioners propose that such rearrangements occur because the c-and zC-type fragment ions are held in close proximity, so that an H atom can be abstracted from the ctype and directed to the zC-type product (this hydrogen-atom transfer occurs prior to the separation of the two fragment ions). [6,7] For large-scale sequencing applications, these rearrangements are problematic, as the mass window needed to define a possible fragment becomes too large.We ...