Molecular and biochemical characterization of key enzymes in the cysteine and serine metabolic pathways of Acanthamoeba castellanii.

BACKGROUND: Acanthamoeba spp. can cause serious human infections, including Acanthamoeba keratitis, granulomatous amoebic encephalitis and cutaneous acanthamoebiasis. Cysteine biosynthesis and the L-serine metabolic pathway play important roles in the energy metabolism of Acanthamoeba spp. However, no study has confirmed the functions of cysteine synthase (AcCS) in the cysteine pathway and phosphoglycerate dehydrogenase (AcGDH) or phosphoserine aminotransferase (AcSPAT) in the non-phosphorylation serine metabolic pathway of Acanthamoeba. METHODS: The AcCS, AcGDH and AcSPAT genes were amplified by PCR, and their recombinant proteins were expressed in Escherichia coli. Polyclonal antibodies against the recombinant proteins were prepared in mice and used to determine the subcellular localisation of each native protein by confocal laser scanning microscopy. The enzymatic activity of each recombinant protein was also analysed. Furthermore, each gene expression level was analysed by quantitative PCR after treatment with different concentrations of cysteine or L-serine. RESULTS: The AcCS gene encodes a 382-amino acid protein with a predicted molecular mass of 43.1 kDa and an isoelectric point (pI) of 8.11. The AcGDH gene encodes a 350-amino acid protein with a predicted molecular mass of 39.1 kDa and a pI of 5.51. The AcSPAT gene encodes a 354-amino acid protein with a predicted molecular mass of 38.3 kDa and a pI of 6.26. Recombinant AcCS exhibited a high cysteine synthesis activity using O-acetylserine and Na2S as substrates. Both GDH and SPAT catalysed degradation, rather than synthesis, of serine. Exogenous L-serine or cysteine inhibited the expression of all three enzymes in a time- and dose-dependent manner. CONCLUSIONS: This study demonstrated that AcCS participates in cysteine biosynthesis and serine degradation via the non-phosphorylation serine metabolic pathway, providing a molecular basis for the discovery of novel anti-Acanthamoeba drugs.

Immunological responses to recombinant cysteine synthase A of Brucella abortus in BALB/c mice.

Brucellosis is a worldwide zoonotic disease. No Brucella vaccine is available for use in humans, and existing animal vaccines have limitations. There is a need to develop a safe and effective vaccine against human and animal brucellosis. In the present study, we generated recombinant cysteine synthase A (rCysK) of Brucella abortus in Escherichia coli and purified it up to homogeneity by metal affinity chromatography. The immunogenicity and protective efficacy of purified rCysK were evaluated in BALB/c mice with Freund's adjuvant, aluminium hydroxide gel or without any adjuvant. High titres of anti-rCysK IgG antibody predominated by IgG1 were observed in all immunized mice. After stimulation with rCysK, the spleen lymphocytes of mice immunized with CysK formulated with aluminium hydroxide gel produced significant levels of IFN-γ. Protection against challenge with virulent B. abortus strain 544 was determined in BALB/c mice, and significant protection was observed in all CysK immunized groups when compared with PBS control. Among all the CysK vaccine groups, comparatively better protection was observed in mice immunized with aluminium hydroxide gel (1.064 units of protection). Overall, the results of the study suggest that rCysK induces primarily Th2 type of immune response and provides partial protection against B. abortus challenge.

Analysis of Streptococcus mutans biofilm proteins recognized by salivary immunoglobulin A.

INTRODUCTION: The purpose of this study was to examine the Streptococcus mutans biofilm cellular proteins recognized by immunoglobulin A (IgA) in saliva from various caries-defined populations. METHODS: Biofilm and planktonic S. mutans UA159 cells were prepared. The proteins were extracted, separated by two-dimensional gel electrophoresis, transferred to blotting membranes, and probed for IgA using individual saliva samples from three groups of subjects; those who developed 0 caries (no active caries), 5-9 caries (medium), or more than 10 caries (severe) over a 12-month interval. RESULTS: Several proteins were recognized by salivary IgA in all groups of saliva but spot distribution and intensity varied greatly between the groups, and some proteins were recognized more strongly in biofilm cells than in planktonic culture, and vice versa. Furthermore, 15 proteins were only recognized by saliva from the 'no active caries' group, and four proteins were recognized by saliva samples from subjects in all three groups. Specifically, antigen I/II was recognized less in biofilm cells by caries-free saliva compared with planktonic cells. However, salivary IgA antibody to antigen I/II was absent in blots using saliva from the 'medium caries' and 'severe caries' groups. CONCLUSION: The bacterial molecules recognized by caries-free saliva are significant factors for S. mutans caries formation, and their inhibition could be a therapeutic target. In addition, saliva of caries-free subjects includes significant IgA antibody against antigen I/II of S. mutans, indicating a protective mechanism. However, microorganisms may protect themselves from host immune attack by forming biofilms and decreasing expression of antigen I/II.

Purification and characterization of O-acetylserine (thiol) lyase from spinach chloroplasts.

O-Acetylserine (thiol) lyase, the last enzyme in the cysteine biosynthetic pathway, was purified to homogeneity from spinach leaf chloroplasts. The enzyme has a molecular mass of 68,000 and consists of two identical subunits of Mr 35,000. The absorption spectrum obtained at pH 7.5 exhibited a peak at 407 nm due to pyridoxal phosphate, and addition of O-acetylserine induced a considerable modification of the spectrum. The pyridoxal phosphate content was found to be 1.1 per subunit of 35,000, and the chromophore was displaced from the enzyme by O-acetylserine, leading to a progressive inactivation of the holoenzyme. Upon gel filtration chromatography on Superdex 200, part of the chloroplastic O-acetylserine (thiol) lyase eluted in association with serine acetyltransferase at a position corresponding to a molecular mass of 310,000 (such a complex called cysteine synthase has been characterized in bacteria). The activity of O-acetylserine (thiol) lyase was optimum between pH 7.5 and 8.5. The apparent Km for O-acetylserine was 1.3 mM and for sulfide was 0.25 mM. The calculated activation energy was 12.6 kcal/mol at 10 mM O-acetylserine. The overall amino-acid composition of spinach chloroplast O-acetylserine (thiol) lyase was different than that determined for the same enzyme (cytosolic?) obtained from a crude extract of spinach leaves. A polyclonal antibody prepared against the chloroplastic O-acetylserine (thiol) lyase exhibited a very low cross-reactivity with a preparation of mitochondrial matrix and cytosolic proteins suggesting that the chloroplastic isoform was distinct from the mitochondrial and cytosolic counterparts.