Phosphoethanolamine Transferase LptA in Haemophilus ducreyi Modifies Lipid A and Contributes to Human Defensin Resistance In Vitro

Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, β-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. The triple mutant was significantly more susceptible to both α- and β-defensins; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin LL-37 may be more important than defensin resistance to H. ducreyi pathogenesis

Identification and characterization of AckA-dependent protein acetylation in <i>Neisseria gonorrhoeae</i>

<div><p><i>Neisseria gonorrhoeae</i>, the causative agent of gonorrhea, has a number of factors known to contribute to pathogenesis; however, a full understanding of these processes and their regulation has proven to be elusive. Post-translational modifications (PTMs) of bacterial proteins are now recognized as one mechanism of protein regulation. In the present study, Western blot analyses, with an anti-acetyl-lysine antibody, indicated that a large number of gonococcal proteins are post-translationally modified. Previous work has shown that N^ε-lysine acetylation can occur non-enzymatically with acetyl-phosphate (AcP) as the acetyl donor. In the current study, an acetate kinase mutant (1291<i>ackA</i>), which accumulates AcP, was generated in <i>N</i>. <i>gonorrhoeae</i>. Broth cultures of <i>N</i>. <i>gonorrhoeae</i> 1291wt and 1291<i>ackA</i> were grown, proteins extracted and digested, and peptides containing acetylated-lysines (K-acetyl) were affinity-enriched from both strains. Mass spectrometric analyses of these samples identified a total of 2686 unique acetylation sites. Label-free relative quantitation of the K-acetyl peptides derived from the <i>ackA</i> and wild-type (wt) strains demonstrated that 109 acetylation sites had an <i>ackA</i>/wt ratio>2 and p-values <0.05 in at least 2/3 of the biological replicates and were designated as “AckA-dependent”. Regulated K-acetyl sites were found in ribosomal proteins, central metabolism proteins, iron acquisition and regulation proteins, pilus assembly and regulation proteins, and a two-component response regulator. Since AckA is part of a metabolic pathway, comparative growth studies of the <i>ackA</i> mutant and wt strains were performed. The mutant showed a growth defect under aerobic conditions, an inability to grow anaerobically, and a defect in biofilm maturation. In conclusion, the current study identified AckA-dependent acetylation sites in <i>N</i>. <i>gonorrhoeae</i> and determined that these sites are found in a diverse group of proteins. This work lays the foundation for future studies focusing on specific acetylation sites that may have relevance in gonococcal pathogenesis and metabolism.</p></div

Characterization of Inner and Outer Membrane Proteins from Francisella tularensis Strains LVS and Schu S4 and Identification of Potential Subunit Vaccine Candidates

Francisella tularensis is the causative agent of tularemia and a potential bioterrorism agent. In the present study, we isolated, identified, and quantified the proteins present in the membranes of the virulent type A strain, Schu S4, and the attenuated type B strain, LVS (live vaccine strain). Spectral counting of mass spectrometric data showed enrichment for membrane proteins in both strains. Mice vaccinated with whole LVS membranes encapsulated in poly (lactic-co-glycolic acid) (PLGA) nanoparticles containing the adjuvant polyinosinic-polycytidylic acid [poly(I·C)] showed significant protection against a challenge with LVS compared to the results seen with naive mice or mice vaccinated with either membranes or poly(I·C) alone. The PLGA-encapsulated Schu S4 membranes with poly(I·C) alone did not significantly protect mice from a lethal intraperitoneal challenge with Schu S4; however, this vaccination strategy provided protection from LVS challenge. Mice that received the encapsulated Schu S4 membranes followed by a booster of LVS bacteria showed significant protection with respect to a lethal Schu S4 challenge compared to control mice. Western blot analyses of the sera from the Schu S4-vaccinated mice that received an LVS booster showed four immunoreactive bands. One of these bands from the corresponding one-dimensional (1D) SDS-PAGE experiment represented capsule. The remaining bands were excised, digested with trypsin, and analyzed using mass spectrometry. The most abundant proteins present in these immunoreactive samples were an outer membrane OmpA-like protein, FopA; the type IV pilus fiber building block protein; a hypothetical membrane protein; and lipoproteins LpnA and Lpp3. These proteins should serve as potential targets for future recombinant protein vaccination studies

Western blots with (A) MAb Acetyl-K(103) and (B) MAb 2C3 loading control.

<p>Samples were taken from broth-grown cultures of 1291wt and 1291<i>ackA</i> at 3, 6, and 24h. The MAb Acetyl-K(103) blot shows that acetylation levels in 1291<i>ackA</i> are higher at every time-point compared to its parent strain 1291wt. The 2C3 blot confirms that equivalent levels of protein were used in these experiments.</p

Detected acetylation sites were mapped onto the crystal structures that had the highest homology to the gonococcal iron binding and iron regulation proteins HemO and Fur.

<p>(A) Crystal structure of HemO from <i>N</i>. <i>meningitidis</i> (PDB: 1P3T). Possible heme binding sites and the putative O₂ activation site (R98) are shown in green. The distance between the predicted O₂ activation site (R98) and the closest AckA-dependent K-acetyl site (K75) was measured to be 16.5 Å. (B) Crystal structure of Fur from <i>V</i>. <i>cholera</i> (PDB: 2W57). A proposed DNA-binding site (residues 1–84) is shown in green. All three of the common K-acetyl sites mapped to the predicted DNA-binding region of Fur. In both panels K-acetyl sites shown in red were found to be AckA-dependent, and all K-acetyl sites shown in blue were detected in the current study. All residue positions of common K sites are listed as their position in their respective <i>N</i>. <i>gonorrhoeae</i> proteins. Protein databank (PDB) identifications are listed for each structure.</p

Biofilm growth comparison.

<p>Stacked z-series of biofilms from various time-points stained with LIVE/DEAD stain (green/red). (A) 1291wt biofilms grown for 6, 24, or 48 h. (B) 1291<i>ackA</i> biofilms grown for 6, 24, or 48 h. These images show that 1291wt is able to form a confluent biofilm that remains viable at 48 h. In contrast, the 1291<i>ackA</i> mutant is initially able to form a biofilm, but by 24 h it is no longer viable. (C) The volume of the biofilms were also determined and analyzed using a Mann-Whitney comparison. A significant difference between overall size of the 1291wt and 1291<i>ackA</i> biofilms were seen at 24 h and 48 h (with p-values of 0.048 and 0.016, respectively). Biofilm experiments were performed in duplicate in two independent experiments.</p

Growth comparisons of 1291wt (■) and 1291<i>ackA</i> (●) from aerobically grown broth cultures.

<p>Samples were taken at 0, 1, 2, 3, 4, 5, 15, and 24 h, plated in triplicate, and the average colony forming units (CFU) determined for each time-point. Duplicate experiments were performed and the results presented are the average of those two values. As can be seen, aerobic growth of 1291<i>ackA</i> is limited in liquid media compared to its parent strain 1291wt.</p

Mass spectrometric workflow.

<p>(A) Three biological replicates of <i>N</i>. <i>gonorrhoeae</i> strains 1291wt and 1291<i>ackA</i> were grown overnight in broth cultures. Cells were harvested, washed, and a protein lysate was generated and digested with trypsin. Affinity enrichment for acetyl-lysine (K-acetyl)-containing peptides was performed using a polyclonal anti-acetyl-Lys antibody. Enriched K-acetyl peptides were analyzed by high-resolution label-free LC-MS/MS and quantified using MS1 quantitation. (B) An example of a peptide analyzed by MS1 quantitation using Skyline. MS1 ion chromatograms of the precursor ions for M, M+1, and M+2 for the acetylated peptide IKLD<u><b>K*</b></u>LVSEGFER, at K-329, from the zinc-binding alcohol dehydrogenase protein for 1291wt and 1291<i>ackA</i> are shown. MS1 quantitation was performed by extracting and measuring the peak areas from the most intense isotopic precursors and subsequently comparing the wt and <i>ackA</i> mutant peptides.</p

Analysis of acetylation sites detected in the present study.

<p>A total of 2686 unique acetylation sites from 656 unique acetylated proteins were observed in the current study. Venn diagrams showing the overlap of 1291wt and 1291<i>ackA</i> (A) acetylation sites and (B) acetylated proteins detected in this study. (C) The number of acetylation sites detected per protein was graphed. (D) Lysine acetylation sites that were observed in two or more biological replicates with a 2-fold or greater <i>ackA</i>/wt ratio and a p-value of <0.05 were designated as “AckA-dependent”. The fold change distribution for these 109 “AckA-dependent” acetylation sites from 70 proteins was then graphed. (E-F) IceLogo generated outputs showing the analysis from the +/-10 amino acids relative to the acetylation sites from all “AckA-dependent” sites. In the heatmap output (E) the color schema generated shows the p-value score for significant amino acid positions. Amino acids highlighted in red indicate that this position is less likely to occur, and those highlighted in green are 99% more likely to occur. In the percentage difference output (F), the amino acids shown occur in that position with a significantly higher frequency than what would be expected based on the amino acid frequency in the whole proteome reference set. The larger the letter the higher the preference is for a particular position in the AckA-dependent motifs.</p

Bacterial strains and plasmids used in study.

<p>^a StrepR, resistance to streptomycin; Cm^R, resistance to chloramphenicol; AmpR, resistance to ampicillin; KanR, resistance to kanamycin; SpecR, resistance to spectinomycin.</p><p>Bacterial strains and plasmids used in study.</p