Identification, Cloning, and Expression of the CAMP factor gene (cfa) of Group A Streptococci

ABSTRACT The CAMP reaction is a synergistic lysis of erythrocytes by the interaction of an extracellular protein (CAMP factor) produced by some streptococcal species with the Staphylococcus aureussphingomyelinase C (beta-toxin). Group A streptococci (GAS [Streptococcus pyogenes]) have been long considered CAMP negative, and this reaction commonly has been used to distinguish GAS from Streptococcus agalactiae. We here provide evidence that GAS possess this gene and produce an extracellular CAMP factor capable of participating in a positive CAMP reaction. The S. pyogenes CAMP factor is specified by a 774-bp open reading frame homologous to the CAMP factor genes from S. agalactiae andStreptococcus uberis. This gene, designatedcfa, was isolated on a 1,256-bp fragment and cloned inEscherichia coli. Recombinant clones of E. coliexpressing cfa secreted an active CAMP factor. The deduced 28.5-kDa protein encoded by cfa consists of 257 amino acids, with a predicted 28-amino-acid signal peptide. Thecfa gene is widely spread among GAS: 82 of 100 clinical GAS isolates produced a positive CAMP reaction. Of the CAMP-negative strains, 17 of the 18 GAS strains contained the cfa gene. Additionally, CAMP activity was detected in streptococci from serogroups C, M, P, R, and U. The cfa gene was cloned and actively expressed in Escherichia coli and gene fusions were made, placing the β-galactosidase gene (lacZ) under control of the cfa promoter. These cfapromoter-lacZ fusions were introduced into S. pyogenes via a bacteriophage-derived site-specific integration vector where they showed that the cfa gene has a strong promoter that may be subject to as-yet-unidentified regulatory factors. The results presented here, along with previous reports, indicate that the CAMP factor gene is fairly widespread among streptococci, being present at least in groups A, B, C, G, M, P, R, and U.

[1]  N. Figura,et al.  Differentiation of motile and mesophilic Aeromonas strains into species by testing for a CAMP-like factor , 1987, Journal of clinical microbiology.

[2]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[3]  M. Gilmore,et al.  PCR amplification of streptococcal DNA using crude cell lysates. , 1992, FEMS microbiology letters.

[4]  R. Möllby,et al.  Phenomenon of hot-cold hemolysis: chelator-induced lysis of sphingomyelinase-treated erythrocytes , 1975, Infection and immunity.

[5]  G. Wiseman,et al.  The nature of staphylococcal beta hemolysin. I. Mode of action. , 1967, Canadian journal of microbiology.

[6]  F. Fehrenbach,et al.  Unspecific binding of group B streptococcal cocytolysin (CAMP factor) to immunoglobulins and its possible role in pathogenicity , 1987, The Journal of experimental medicine.

[7]  G. Fraser.  Hæmolytic Activity of Corynebacterium ovis , 1961, Nature.

[8]  W. Köhler.  CAMP-like phenomena of Vibrios. , 1988, Zentralblatt fur Bakteriologie, Mikrobiologie, und Hygiene. Series A, Medical microbiology, infectious diseases, virology, parasitology.

[9]  B. Skalka,et al.  Lethal effect of CAMP-factor and UBERIS-factor--a new finding about diffusible exosubstances of streptococcus agalactiae and Streptococcus uberis. , 1981, Zentralblatt fur Bakteriologie. 1. Abt. Originale. A: Medizinische Mikrobiologie, Infektionskrankheiten und Parasitologie.

[10]  S. M. Gubash.  Synergistic hemolysis phenomenon shown by an alpha-toxin-producing Clostridium perfingens and streptococcal CAMP factor in presumptive streptococcal grouping , 1978, Journal of clinical microbiology.

[11]  D. Smith,et al.  Rapid tube CAMP test for identification of Streptococcus agalactiae (Lancefield group B) , 1980, Journal of clinical microbiology.

[12]  L. Babiuk,et al.  Cloning, sequencing and expression of the CAMP factor gene of Streptococcus uberis. , 1996, Microbial pathogenesis.

[13]  J. Ferretti,et al.  Electrotransformation of Streptococcus pyogenes with plasmid and linear DNA. , 1991, FEMS microbiology letters.

[14]  J. Wecke,et al.  Immunochemical localization of CAMP factor (protein B) in Streptococcus agalactiae. , 1997, Microbios.

[15]  K. Gase,et al.  Cloning, sequencing and functional overexpression of the Streptococcus equisimilis H46A gapC gene encoding a glyceraldehyde-3-phosphate dehydrogenase that also functions as a plasmin(ogen)-binding protein. Purification and biochemical characterization of the protein. , 1996, European journal of biochemistry.

[16]  P. Grimont,et al.  Notes: Listeria welshimeri sp. nov. and Listeria seeligeri sp. nov. , 1983 .

[17]  F. Richaud,et al.  New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacZ gene sequences encoding an enzymatically active carboxy-terminal portion of beta-galactosidase. , 1983, Gene.

[18]  A. W. Bernheimer,et al.  Nature and mechanism of action of the CAMP protein of group B streptococci , 1979, Infection and immunity.

[19]  J. Alouf.  Bacterial Protein Toxins , 2000 .

[20]  C. L. Darling.  Standardization and evaluation of the CAMP reaction for the prompt, presumptive identification of Streptococcus agalactiae (Lancefield group B) in clinical material , 1975, Journal of clinical microbiology.

[21]  W. McClure,et al.  Mechanism and control of transcription initiation in prokaryotes. , 1985, Annual review of biochemistry.

[22]  O. Schneewind,et al.  Cloning and expression of the CAMP factor of group B streptococci in Escherichia coli , 1988, Infection and immunity.

[23]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[24]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[25]  J. Ferretti,et al.  Vectors containing streptococcal bacteriophage integrases for site-specific gene insertion , 1998 .

[26]  J. Nicolet,et al.  Cloning and expression of a cohemolysin, the CAMP factor of Actinobacillus pleuropneumoniae , 1989, Infection and immunity.

[27]  D. Worrall,et al.  The formation of biologically active beta-galactosidase inclusion bodies in Escherichia coli. , 1989, Australian journal of biotechnology.

[28]  S. Brunak,et al.  SHORT COMMUNICATION Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites , 1997 .

[29]  D. Leach,et al.  A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria . By Jeffrey H. Miller. Cold Spring Harbor Laboratory Press. 1992. 876 pages. Price $95.00. ISBN 0 87969 349 5. , 1993 .

[30]  David J. States,et al.  Identification of protein coding regions by database similarity search , 1993, Nature Genetics.

[31]  N. Saunders,et al.  Rapid extraction of bacterial genomic DNA with guanidium thiocyanate , 1989 .

[32]  C. Lämmler,et al.  Comparative studies on streptococci of serological group G isolated from various origins. , 1996, Zentralblatt fur Veterinarmedizin. Reihe B. Journal of veterinary medicine. Series B.

[33]  Physical map location of the new artPIQMJ genes of Escherichia coli, encoding a periplasmic arginine transport system , 1993, Journal of bacteriology.

[34]  F. Fehrenbach,et al.  Reaction components influencing CAMP factor induced lysis. , 1985, Journal of general microbiology.

[35]  S. Fisher,et al.  Expression of the Bacillus subtilis gabP gene is regulated independently in response to nitrogen and amino acid availability , 1996, Molecular microbiology.

[36]  J. Ferretti,et al.  A physical and genetic map of an M type 1 strain of Streptococcus pyogenes. , 1995, Developments in biological standardization.

[37]  C. Steinberg,et al.  Influence of PCBs on the predator-prey relation between bacteria and protozoa in soil , 1990 .

[38]  G. Fraser.  The Haemolysis of Animal Erythrocytes by Pasteurella haemolytica Produced in Conjunction with Certain Staphylococcal Toxins , 1962 .

[39]  J. Ferretti,et al.  Bacteriophage T12 of Streptococcus pyogenes integrates into the gene encoding a serine tRNA , 1997, Molecular microbiology.

[40]  L. Babiuk,et al.  Cloning, sequencing and expression of the CAMP factor gene of Streptococcus uberis. , 1996, Microbial pathogenesis.

[41]  H. Wilkinson.  CAMP-disk test for presumptive identification of group B streptococci , 1977, Journal of clinical microbiology.

[42]  J. Tapsall,et al.  Streptococcus pyogenes streptolysin O as a cause of false-positive CAMP reactions , 1984, Journal of clinical microbiology.

[43]  Worrall Dm,et al.  The formation of biologically active beta-galactosidase inclusion bodies in Escherichia coli. , 1989 .

[44]  E. Munch-Petersen,et al.  A NOTE ON A LYTIC PHENOMENON SHOWN BY GROUP B STREPTOCOCCI , 1944 .

[45]  C. Yanisch-Perron,et al.  Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. , 1985, Gene.

[46]  Anthony Maxwell,et al.  Mode of Action , 1998 .