Generation and characterization of an inter-generic bivalent alpha domain fusion protein αCS from Clostridium perfringens and Staphylococcus aureus for concurrent diagnosis and therapeutic applications

Aim: To evaluate an inter‐generic recombinant alpha domain fusion protein for simultaneous detection and neutralization of Clostridium perfringens and Staphylococcus aureus alpha toxins. Methods and Results: Truncated portions of clostridial and staphylococcal alpha haemolysin genes were PCR amplified and linked to each other through a hydrophilic flexible Glycine linker sequence using overlap‐extension PCR to form a chimeric gene αCS. The recombinant αCS fusion protein was expressed and characterized for its toxicity, cell binding capacity and haemolysis inhibition properties. The fusion protein was nontoxic and effectively retarded staphylococcal alpha haemolysis, probably by competitively interacting with putative staphylococcal alpha haemolysin receptors on erythrocytes. Murine hyperimmune polysera raised against r‐αCS specifically detected 42‐kDa and 33‐kDa proteins when culture supernatants of Cl. perfringens (clostridial alpha toxin) and Staph. aureus (staphylococcal alpha toxin), respectively, were analysed in Western blot. The polyclonal antisera effectively diminished the haemolytic action of both the wild‐type toxins in vitro. Conclusions: The r‐αCS fusion protein was nontoxic competitive inhibitor of staphylococcal alpha haemolysin. The protein elicited specific immune response against Cl. perfringens and Staph. aureus alpha toxins. The antisera also neutralized the toxicities of both the native wild‐type toxins in vitro. Significance of the Study: The bivalent recombinant αCS protein could be a novel intervention in the field of diagnostics and therapeutics against Cl. perfringens and Staph. aureus infections, particularly, in case of co‐infections like gangrenous ischaemia, gangrenous mastitis, etc.     

Molecular Cloning and Characterization of cgs, the Brucella abortus Cyclic β(1-2) Glucan Synthetase Gene: Genetic Complementation of Rhizobium meliloti ndvB and Agrobacterium tumefaciens chvB Mutants

The animal pathogen Brucella abortus contains a gene, cgs, that complemented a Rhizobium meliloti nodule development (ndvB) mutant and an Agrobacterium tumefaciens chromosomal virulence (chvB) mutant. The complemented strains recovered the synthesis of cyclic β(1-2) glucan, motility, virulence in A. tumefaciens, and nitrogen fixation in R. meliloti; all traits were strictly associated with the presence of an active cyclic β(1-2) glucan synthetase protein in the membranes. Nucleotide sequencing revealed the presence in B. abortus of an 8.49-kb open reading frame coding for a predicted membrane protein of 2,831 amino acids (316.2 kDa) and with 51% identity to R. meliloti NdvB. Four regions of the B. abortus protein spanning amino acids 520 to 800, 1025 to 1124, 1284 to 1526, and 2400 to 2660 displayed similarities of higher than 80% with R. meliloti NdvB. Tn3-HoHo1 mutagenesis showed that the C-terminal 825 amino acids of the Brucella protein, although highly conserved in Rhizobium, are not necessary for cyclic β(1-2) glucan synthesis. Confirmation of the identity of this protein as B. abortus cyclic β(1-2) glucan synthetase was done by the construction of a B. abortus Tn3-HoHo1 insertion mutant that does not form cyclic β(1-2) glucan and lacks the 316.2-kDa membrane protein. The recovery of this mutant from the spleens of inoculated mice was decreased by 3 orders of magnitude compared with that of the parental strain; this result suggests that cyclic β(1-2) glucan may be a virulence factor in Brucella infection.     

The RTX haemolysins ApxI and ApxII are major virulence factors of the swine pathogen Actinobacillus pleuropneumoniae: evidence from mutational analysis

The involvement of the RTX haemolysins (Apxl and ApxII) of the swine pathogen Actinobacillus pleuropneumoniae in virulence was investigated using haemolysin-deficient mutants constructed by a mini-Tn10 mutagenesis procedure. Two types of haemolysin mutant with single insertions of the transposon were obtained from a serotype 1 strain producing both ApxI and ApxII. One presented a complete loss of haemolytic activity because of the absence of ApxI and ApxII production. The other displayed weaker haemolysis than the wild type and produced only ApxII. The chromosomal regions flanking mini-Tn10 were cloned and sequenced. In the non-haemolytic mutant, the transposon had inserted in apxIB, a gene involved in the exportation of ApxI and ApxII toxins. The weakly haemolytic mutant resulted from the disruption of the structural gene for ApxI. Both mutations In the apxI operon were associated with a significant loss of virulence for mice and pigs, demonstrating that haemolysins are involved in A. pleuropneumoniae pathogenicity. The non-haemolytic mutant was apathogenic and the weakly haemolytic mutant retained some virulence for pigs, suggesting that both ApxI and ApxII are needed for full virulence.     

The auxiliary protein complex SaePQ activates the phosphatase activity of sensor kinase SaeS in the SaeRS two‐component system of Staphylococcus aureus

In bacterial two‐component regulatory systems (TCSs), dephosphorylation of phosphorylated response regulators is essential for resetting the activated systems to the pre‐activation state. However, in the SaeRS TCS, a major virulence TCS of Staphylococcus aureus, the mechanism for dephosphorylation of the response regulator SaeR has not been identified. Here we report that two auxiliary proteins from the sae operon, SaeP and SaeQ, form a protein complex with the sensor kinase SaeS and activate the sensor kinase's phosphatase activity. Efficient activation of the phosphatase activity required the presence of both SaeP and SaeQ. When SaeP and SaeQ were ectopically expressed, the expression of coagulase, a sae target with low affinity for phosphorylated SaeR, was greatly reduced, while the expression of alpha‐haemolysin, a sae target with high affinity for phosphorylated SaeR, was not, demonstrating a differential effect of SaePQ on sae target gene expression. When expression of SaePQ was abolished, most sae target genes were induced at an elevated level. Since the expression of SaeP and SaeQ is induced by the SaeRS TCS, these results suggest that the SaeRS TCS returns to the pre‐activation state by a negative feedback mechanism.     

Yersinia pestis pH 6 antigen forms fimbriae and is induced by intracellular association with macrophages

Ability to express pH 6 antigen (Ag) is necessary for full virulence of Yersinia pestis; however, the function of the Ag in pathogenesis remains unclear. We determined the nucleotide sequence of a 4232 bp region of Y. pestis DNA which encoded the pH 6 Ag structural gene (psaA) and accessory loci necessary for Ag synthesis. Protein sequences encoded by the Y. pestis DNA were similar to accessory proteins which function in the biosynthesis of Escherichia coli fimbriae Pap, K88, K99 and CS3 as well as the molecular chaperone for the Y. pestis capsule protein. Electron microscopy and immunogold labelling studies revealed that pH 6 Ag expressing E coU or Yersinia produced flexible‘fibrillar’organelles composed of individual linear strands, multiple strand bundles or wiry aggregates of PsaA. Y. pestis associated with the murine macrophage-like cell line, RAW264.7, expressed pH 6 Ag in an intracellular acidification-dependent manner. Together with an earlier study showing that a Y. pestis psaA mutant was reduced in virulence, these results demonstrate that the expression of fimbriae which are induced in host macrophages is involved in plague pathogenesis.     

Induction of haemolytic activity in Escherichia coli by the slyA gene product

The Salmonella typhimurium protein SlyA_ST, originally described as a cytolysin, shows sequence similarities to several known bacterial regulatory proteins. A homologue to the slyA_St gene has been localised to min 37 of the Eschericia coli K-12 chromosome and has been designated slyA_EC When introduced in trans on a plasmid, the slyA_EC gene conferred a haemolytic phenotype on wild-type but not clyA-knockout strains of E. coli K-12. The clyA gene encodes a novel haemolysin that is not expressed by wild-type E. coli under tested laboratory conditions. Western and Northern blot analyses, and DNA-band-shift assays support a model whereby the SlyA_EC protein activates clyA expression by binding to the clyA promoter region, thereby supporting the sequence similarity data in suggesting that SlyA_ST is a haemolysin activator rather than being a haemolysin per se.     

Evidence for autolysin-mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface

Biofilm formation on a polymer surface which involves initial attachment and accumulation in multilayered cell clusters (intercellular adhesion) is proposed to be the major pathogenicity factor in Staphylococcus epidermidis foreign-body-associated infections. We have characterized two distinct classes of biofilm-negative Tn917 mutants in S. epidermidis affected in initial attachment (class A) or intercellular adhesion (class B). mut1 (class A mutant) lacks five surface-associated proteins with molecular masses of 120, 60, 52, 45 and 38 kDa and could be complemented by transformation with a 16.4 kb wild-type DNA fragment. The complemented mutant was able to attach to a polystyrene surface, to form a biofilm, and produced all of the proteins missing from mut1. Subcloning experiments revealed that the 60 kDa protein is sufficient for initial attachment. Immunofluorescence microscopy using an antiserum raised against the 60 kDa protein showed that this protein is located at the cell surface. DNA-sequence analysis of the complementing region revealed a single open reading frame which consists of 4005 nucleotides and encodes a deduced protein of 1335 amino acids with a predicted molecular mass of 148 kDa. The amino acid sequence exhibits a high similarity (61% identical amino acids) to the atl gene product of Staphylococcus aureus, which represents the major autolysin; therefore the open reading frame was designated atlE. By analogy with the S. aureus autolysin, AtlE is composed of two bacteriolytically active domains, a 60 kDa amidase and a 52 kDa glucosaminidase domain, generated by proteolytic processing. The 120 kDa protein missing from mut1 presumably represents the unprocessed amidase and glucosaminidase domain after proteolytic cleavage of the signal- and propeptide. The 45 and 38 kDa proteins are probably the degradation products of the 60 and 52 kDa proteins, respectively. Additionally, AtlE was found to exhibit vitronectin-binding activity, indicating that AtlE plays a role in binding of the cells not only to a naked polystyrene surface during early stages of adherence, but also to plasma protein-coated polymer surfaces during later stages of adherence. Our findings provide evidence for a new function of an autolysin (AtlE) in mediating the attachment of bacterial cells to a polymer surface, representing the prerequisite for biofilm formation.     

Chrysin protects mice from Staphylococcus aureus pneumonia

Aim: To elucidate the effect of chrysin on α‐haemolysin production by Staphylococcus aureus and protection against pneumonia in a murine model. Methods and Results: Haemolysis, Western blot and real‐time RT‐PCR assays were performed to evaluate the effect of chrysin on α‐haemolysin secretion by Staph. aureus. The efficacy of chrysin against human alveolar epithelial cell injury by α‐haemolysin was tested using live/dead staining or by measuring lactate dehydrogenase activity. Furthermore, we determined the protective effect of chrysin against Staph. aureus pneumonia through histopathology experiments in a mouse model. The production of α‐haemolysin by Staph. aureus was inhibited when presented with an increasing subinhibitory concentration of chrysin in vitro. Consistent with this result, chrysin prevented α‐haemolysin‐mediated cell injury and protected mice from Staph. aureus pneumonia. Conclusions: Chrysin is a potent inhibitor of α‐haemolysin expression by Staph. aureus, and it conferred a significant degree of protection against Staph. aureus pneumonia. Significance and Impact of Study: The chrysin‐mediated inhibition of α‐haemolysin production and protection against Staph. aureus pneumonia may offer a new strategy in combating pathogen infections.     

Expression and Purification of Recombinant lysostaphin in Escherichia coli

Summary A 1.5 kb plasmid-encoded lysostaphin gene fragment of Staphylococcus staphylolyticus was amplified by polymerase chain reaction (PCR) and cloned in Escherichia coli by using plasmid pET29b(+) as an expression vector. By optimizing culture conditions, the activities of lysostaphin were expressed as 66 %, 30 %, and 4 % in extracellular, intracellular, and periplasmic fractions of recombinant E. coli, respectively. The enzyme was purified to homogeneity by using a simple one-step fractionation on bacterial cells of lysostaphin-resistant Staphylococcus aureus mutant. The recombinant enzyme had an Mr of approximate 27 kDa, and its bacteriolytic activity was indistinguishable to the authentic lysostaphin purified from Staphylococcus staphylolyticus.     

Expression of the pspA gene stimulates efficient protein export in Escherichia coli

Expression of several mutant forms of outer membrane protein PhoE of Escherichia coli, which are disturbed in normal biogenesis, resulted in high expression of a 26kDa protein. This 26kDa protein fractionated as a peripherally bound inner membrane protein. It appeared to be identical to a previously identified protein (PspA = phage shock protein A) of unknown function that is induced upon infection of E. coli with filamentous phages. PspA was not expressed upon synthesis of mutant PhoE proteins in a secB mutant, nor upon expression of a PhoE mutant that lacks the signal sequence, suggesting that entrance into the export pathway of prePhoE is essential for induction. PspA synthesis was also induced under other conditions that are known to block the export apparatus, i.e. in secA, secD and secF mutants when grown at their non-permissive temperature or upon induction of the synthesis of MalE-LacZ or LamB-LacZ hybrid proteins. The inducing conditions for PspA synthesis suggested a rote for this protein in export. In vivo pulse-chase experiments showed that the translocation of (mutant) prePhoE and of the precursors of other exported proteins was retarded in a pspA mutant strain. Also, in in vitro translocation assays, a role for PspA in protein transport could be demonstrated.     

Cloning and characterization of a phospholipase gene from Erwinia chrysanthemi EC16.

A single gene (plcA) was cloned from a cosmid library of Erwinia chrysanthemi EC16 DNA that encoded an extracellular phospholipase. The gene was subcloned and DNA sequence data showed the presence of a single open reading frame encoding a protein with a predicted size of 39kDa. The coding region was G+C-rich and the protein had a predicted basic isoelectric point. The protein showed no significant homology with others in the PIR library, including other phospholipases. When overexpressed in Escherichia coli cells, the plcA gene directed production of a c. 39kDa protein that was largely localized in the periplasm, but its N-terminal amino acid sequence was that of the native protein predicted from DNA sequence data. Unlike the wild-type bacterium, an E. chrysanthemi EC16 marker exchange mutant of the plcA gene did not secrete extracellular phospholipase activity into the medium. However, no detectable change was observed in terms of the virulence of the mutant strain on potato tubers or chrysanthemum stems.