Bacterial and Arachnid Sphingomyelinases D: Comparison of Biophysical and Pathological Activities.

Sphingomyelinases D have only been identified in arachnid venoms, Corynebacteria, Arcanobacterium, Photobacterium and in the fungi Aspergillus and Coccidioides. The arachnid and bacterial enzymes share very low sequence identity and do not contain the HKD sequence motif characteristic of the phospholipase D superfamily, however, molecular modeling and circular dichroism of SMases D from Loxosceles intermedia and Corynebacterium pseudotuberculosis indicate similar folds. The phospholipase, hemolytic and necrotic activities and mice vessel permeabilities were compared and both enzymes possess the ability to hydrolyze phospholipids and also promote similar pathological reactions in the host suggesting the existence of a common underlying mechanism in tissue disruption. J. Cell. Biochem. 118:2053-2063, 2017. © 2016 Wiley Periodicals, Inc.

Tyrosine binding and promiscuity in the arginine repressor from the pathogenic bacterium Corynebacterium pseudotuberculosis.

The arginine repressor (ArgR) regulates arginine biosynthesis in a number of microorganisms and consists of two domains interlinked by a short peptide; the N-terminal domain is involved in DNA binding and the C-terminal domain binds arginine and forms a hexamer made-up of a dimer of trimers. The crystal structure of the C-terminal domain of ArgR from the pathogenic Corynebacterium pseudotuberculosis determined at 1.9 Å resolution contains a tightly bound tyrosine at the arginine-binding site indicating hitherto unobserved promiscuity. Structural analysis of the binding pocket displays clear molecular adaptations to accommodate tyrosine binding suggesting the possible existence of an alternative regulatory process in this pathogenic bacterium.

CP40 from Corynebacterium pseudotuberculosis is an endo-ß-N-acetylglucosaminidase.

BACKGROUND: C. pseudotuberculosis is an important animal pathogen that causes substantial economical loss in sheep and goat farming. Zoonotic infections in humans are rare, but when they occur they are often severe and difficult to treat. One of the most studied proteins from this bacterium, the secreted protein CP40 is being developed as a promising vaccine candidate and has been characterized as a serine protease. In this study we have investigated if CP40 is an endoglycosidase rather than a protease. RESULTS: CP40 does not show any protease activity and contains an EndoS-like family 18 of glycoside hydrolase (chitinase) motif. It hydrolyzes biantennary glycans on both human and ovine IgGs. CP40 is not a general chitinase and cannot hydrolyze bisecting GlcNAc. CONCLUSION: Taken together we present solid evidence for re-annotating CP40 as an EndoS-like endoglycosidase. Redefining the activity of this enzyme will facilitate subsequent studies that could give further insight into immune evasion mechanisms underlying corynebacterial infections in animals and humans.

Identification of 11 new exoproteins in Corynebacterium pseudotuberculosis by comparative analysis of the exoproteome.

This study involves the comparison between the exoproteomes of two different strains of Corynebacterium pseudotuberculosis, the etiologic agent of caseous lymphadenitis in small ruminants. In a previous study, based on a gel-free system (TPP-LC/MS(E)), 70 exoproteins for the strain 1002 and 67 for the strain C231, totaling 93 different extracellular proteins for C. pseudotuberculosis, were identified. In the present work, we have used 2D gel electrophoresis to resolve the extracellular proteins of both strains, which were then digested with trypsin, analyzed by MALDI-TOF/TOF and identified with the software MASCOT(®). A total of 45 extracellular proteins of C. pseudotuberculosis were identified by this approach. The comparative analysis between the strains 1002 and C231 identified 13 and 3 strain-specific proteins, respectively, 11 of which are novel. These newly identified proteins may play an important role in the physiology and virulence of C. pseudotuberculosis.

Differential Exoproteome analysis of two corynebacterium pseudotuberculosis biovar ovis strains isolated from goat (1002) and sheep (C231).

Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis a chronic infectious disease affecting small ruminants. The 2D-DIGE technique was used to compare the exoproteomes of two C. pseudotuberculosis biovar ovis strains isolated from goat (strain 1002) and sheep (strain C231). Seventeen proteins differentially produced were identified here. Nine proteins appeared over-produced in the exoproteome of 1002 goat strain and 8 in that of C231 sheep strain. These proteins were related to various biological functions, such as the cell envelope, respiratory metabolism and proteolysis. This proteomic analysis revealed strain-specific exoproteins although each of the corresponding genes was found in both strain genomes. Such differential expression pattern may reflect inter-strain differences in adaptation to a specific host, in pathogenicity and or in antigenicity of this pathogenic bacterium.

Cell wall glycolipids from Corynebacterium pseudotuberculosis strains with different virulences differ in terms of composition and immune recognition.

Caseous lymphadenitis (CLA) is an infectious disease caused by Corynebacterium pseudotuberculosis in small ruminants and is characterized by the development of granulomas in the lymph nodes, spleen, liver, and lungs. Although little is known about the host-pathogen relationship of this bacterium, it was previously reported that the pathogen's lipids are important for its taxonomic classification and survival inside macrophages. However, there are no studies regarding the composition of these molecules. In this study, cell wall glycolipids from two C. pseudotuberculosis strains presenting different virulence profiles were purified and its composition was characterized. A difference was observed between the electrophoretic and chromatogram profiles for cell wall components from the two strains, mainly among molecules with low molecular weights. IgM from sheep with acute CLA recognized antigens with an estimated molecular weight of 11 kDa of the low-pathogenicity strain, while low-molecular weight antigens from the high-pathogenicity strain presented a lower recognition by these antibodies. Mass spectrometry analysis showed that the cell wall of the high-pathogenicity strain contained glycolipids with high amounts of unsaturated fatty acids and glycerophosphoinositols, which may contribute to the capacity of this strain to cause severe disease. In conclusion, it is indicated that cell wall non-protein antigens can play a key role in C. pseudotuberculosis virulence.

Association of Corynebacterium pseudotuberculosis recombinant proteins rCP09720 or rCP01850 with rPLD as immunogens in caseous lymphadenitis immunoprophylaxis.

Caseous lymphadenitis (CLA) is a chronic disease responsible for significant economic losses in sheep and goat breeding worldwide. The treatment for this disease is not effective, and an intense vaccination schedule would be the best control strategy. In this study, we evaluated the associations of rCP09720 or rCP01850 proteins from Corynebacterium pseudotuberculosis with recombinant exotoxin phospholipase D (rPLD) as subunit vaccines in mice. Four experimental groups (10 animals each) were immunized with a sterile 0.9% saline solution (G1), rPLD (G2), rPLD + rCP09720 (G3), and rPLD + rCP01850 (G4). The mice received two doses of each vaccine at a 21-day interval and were challenged 21 days after the last immunization. The animals were evaluated daily for 40 days after the challenge, and mortality rate was recorded. The total IgG production level increased significantly in the experimental groups on day 42 after the first vaccination. Similarly, higher levels of specific IgG2a were observed in experimental groups G2, G3, and G4 compared to the IgG1 levels on day 42. G4 showed a significant (p < .05) humoral response against both antigens of the antigenic formulations. The cellular immune response induced by immunization was characterized by a significant (p < .05) production of interferon-γ compared to that in the control, while the concentrations of interleukin (IL)-4 and IL-12 were not significant in any group. A significant increase of tumor necrosis factor was observed only in G4. The survival rates after the challenge were 30% (rPLD), 40% (rPLD + rCP09720), and 50% (rPLD + rCP01850). Thus, the association of rCP01850 with rPLD resulted in the best protection against the challenge with C. pseudotuberculosis and induced a more intense type 1 T-helper cell immune response.

Cold Shock Protein A from Corynebacterium pseudotuberculosis: Role of Electrostatic Forces in the Stability of the Secondary Structure.

The conformational stability of the Cold shock protein A (CspA) from C. pseudotuberculosis (Cp), a nucleic acid binding protein in function of pH and salt concentration was examined by using differential scanning calorimetry and CD spectroscopy in combination with computational analysis to identify the specify amino acids undergoing change. Our approach identified a sodiumbinding site in CpCspA and at pH 8.0 a significant reduction in the ß-sheet content was observed which resulted in a decrease of the protein thermal stability. The computational analyses identified His30 and His65 as the amino acids with the largest charge shifts at different pHs. His30/His65 are part of the extensive hydrogen bonding network and along with the ion-binding site are essential for the conformational stability of CspA.

Changes in protein abundance are observed in bacterial isolates from a natural host.

Bacterial proteomic studies frequently use strains cultured in synthetic liquid media over many generations. It is uncertain whether bacterial proteins expressed under these conditions will be the same as the repertoire found in natural environments, or when bacteria are infecting a host organism. Thus, genomic and proteomic characterization of bacteria derived from the host environment in comparison to reference strains grown in the lab, should aid understanding of pathogenesis. Isolates of Corynebacterium pseudotuberculosis were obtained from the lymph nodes of three naturally infected sheep and compared to a laboratory reference strain using bottom-up proteomics, after whole genome sequencing of each of the field isolates. These comparisons were performed following growth in liquid media that allowed us to reach the required protein amount for proteomic analysis. Over 1350 proteins were identified in the isolated strains, from which unique proteome features were revealed. Several of the identified proteins demonstrated a significant abundance difference in the field isolates compared to the reference strain even though there were no obvious differences in the DNA sequence of the corresponding gene or in nearby non-coding DNA. Higher abundance in the field isolates was observed for proteins related to hypoxia and nutrient deficiency responses as well as to thiopeptide biosynthesis.

Chromatographic and mass spectrometric characterization of 3-O-benzoyl methyl ester derivatives of mycolic acid fractions from Corynebacterium pseudotuberculosis, C. diphtheriae and Rhodococcus rhodochrous.

A benzoyl group was attached to the 3-hydroxyl group of the methyl ester derivative of corynomycolic acid fraction isolated from Corynebacterium pseudotuberculosis. The infrared spectrum of the 3-O-benzoylated compound displayed a series of characteristic absorptions found at 1110, 1267 and 1603 cm-1 that confirmed the presence of a monosubstituted phenyl grouping. The 1H-NMR spectrum showed peaks representing protons of the aromatic ring at 7.4 ppm and 8.0 ppm. The UV spectrum revealed two absorption maxima: at 190 and 228 nm. The mass spectrum of the 3-O-benzoylated material exhibited the following peaks: (1) a prominent peak at m/z 105 of the benzoyl group that constituted the base peak; (2) peaks of methyl esters representing the alpha-hydrocarbon side chain plus carbon atoms C1 and C2 of the mycolic acid molecule; and (3) peaks of molecular ion minus benzoic acid and/or molecular ion minus benzoxyl group. When subjected to liquid chromatography (LC) on an octadecylsilane-silica gel column the 3-O-benzoylated methyl ester derivatives of the corynomycolic acid fraction were separated into their constituent homologous fractions corresponding to underivatized corynomycolic acids with the chain length C30, C32 and C34. Reversed phase HPLC of saturated and monounsaturated species of 3-O-benzoylated derivatives of the mycolic acid fraction from C. diphtheriae and Rhodococcus rhodochrous led to the separation of the corresponding homologous fractions. Mass spectrometry by electron impact mode identified both series of the homologous materials differing in mass by 28 units.

Characterization of detergent-soluble proteins of Corynebacterium pseudotuberculosis.

Whole cells and culture supernatant of isolates of Corynebacterium pseudotuberculosis were studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (immunoblotting). SDS-PAGE analysis of detergent-solubilized whole cells revealed more than 20 bands in silver-stained gels. However, the SDS-soluble proteins that are present in all the isolates of the bacterium can be separated into four groups, as follows, (i) high molecular mass proteins that are greater than 119 kDa, (ii) a set of three proteins with molecular mass of 84, 64 and 58 kDa, (iii) a doublet consisting of proteins of molecular mass 33 to 30 kDa, and (iv) low molecular mass proteins of 25 to 20 kDa. SDS-PAGE analysis of ammonium sulphate concentrated culture supernatant demonstrated more than seven bands in silver-stained gels ranging in molecular mass from 64-14 kDa. Sera from goats with C. pseudotuberculosis-induced disease were used to probe immunoblots of electrophoresed SDS-soluble proteins. Ten or more SDS-soluble proteins from whole cells, ranging in molecular mass from 119-20 kDa were recognized by antibodies in sera of naturally infected goats. These sera also recognized up to five molecules ranging from 64-30 kDa, on immunoblots of ammonium sulfate concentrated culture supernatant.

Homology modeling, molecular dynamics and QM/MM study of the regulatory protein PhoP from Corynebacterium pseudotuberculosis.

Corynebacterium pseudotuberculosis is a facultatively intracellular Gram-positive bacterium that causes caseous lymphadenitis, principally in sheep and goats, though sometimes in other species of animals, leading to considerable economic losses. This pathogen has a TCS known as PhoPR, which consists of a sensory histidine kinase protein (PhoR) and an intracellular response regulator protein (PhoP). This system is involved in the regulation of proteins present in various processes, including virulence. The regulation is activated by PhoP protein phosphorylation, an event that requires a magnesium (Mg(2+)) ion. Here we describe the 3D structure of the regulatory response protein (PhoP) of C. pseudotuberculosis through molecular modeling by homology. The model generated provides the first structural information on a full-length member of the OmpR/PhoP subfamily. Classical molecular dynamics was used to investigate the stability of the model. In addition, we used quantum mechanical/molecular mechanical techniques to perform (internal, potential) energy optimizations to determine the interaction energy between the Mg(2+) ion and the structure of the PhoP protein. Analysis of the interaction energy residue by residue shows that Asp-16 and Asp-59 play an important role in the protein-Mg(2+) ion interactions. These results may be useful for the future development of a new vaccine against tuberculosis based on genetic attenuation via a point mutation that results in the polar residue Asp-16 and/or Asp-59 being replaced with a nonpolar residue in the DNA-binding domain of PhoP of C. pseudotuberculosis.