ABSTRACT
Type III secretion (T3S) is utilized by a wide range of gram-negative bacterial pathogens to allow the efficient delivery of effector proteins into the host cell cytoplasm through the use of a syringe-like injectisome. Chlamydophila pneumoniae is a gram-negative, obligate intracellular pathogen that has the structural genes coding for a T3S system, but the functionality of the system has not yet been demonstrated. T3S is dependent on ATPase activity, which catalyzes the unfolding of proteins and the secretion of effector proteins through the injectisome. CdsN (Cpn0707) is predicted to be the T3S ATPase of C. pneumoniae based on sequence similarity to other T3S ATPases. Full-length CdsN and a C-terminal truncation of CdsN were cloned as glutathione S-transferase (GST)-tagged constructs and expressed in Escherichia coli. The GST-tagged C-terminal truncation of CdsN possessed ATPase activity, catalyzing the release of ADP and P(i) from ATP at a rate of 0.55 +/- 0.07 micromol min(-1) mg(-1) in a time- and dose-dependent manner. CdsN formed oligomers and high-molecular-weight multimers, as assessed by formaldehyde fixation and nondenaturing polyacrylamide gel electrophoresis. Using bacterial two-hybrid and GST pull-down assays, CdsN was shown to interact with CdsD, CdsL, CdsQ, and CopN, four putative structural components of the C. pneumoniae T3S system. CdsN also interacted with an unannotated protein, Cpn0706, a putative CdsN chaperone. Interactions between CdsN, CdsD, and CopN represent novel interactions not previously reported for other bacterial T3S systems and may be important in the localization and/or function of the ATPase at the inner membrane of C. pneumoniae.
Subject(s)
Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Chlamydophila pneumoniae/enzymology , Chlamydophila pneumoniae/genetics , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/isolation & purification , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Gene Expression , Kinetics , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/isolation & purification , Molecular Sequence Data , Molecular Weight , Phosphorus/metabolism , Protein Binding , Protein Interaction Mapping , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Alignment , Sequence DeletionABSTRACT
Two different severe acute respiratory syndrome (SARS) vaccine strategies were evaluated for their ability to protect against live SARS coronavirus (CoV) challenge in a murine model of infection. A whole killed (inactivated by beta-propiolactone) SARS-CoV vaccine and a combination of two adenovirus-based vectors, one expressing the nucleocapsid (N) and the other expressing the spike (S) protein (collectively designated Ad S/N), were evaluated for the induction of serum neutralizing antibodies and cellular immune responses and their ability to protect against pulmonary SARS-CoV replication. The whole killed virus (WKV) vaccine given subcutaneously to 129S6/SvEv mice was more effective than the Ad S/N vaccine administered either intranasally or intramuscularly in inhibiting SARS-CoV replication in the murine respiratory tract. This protective ability of the WKV vaccine correlated with the induction of high serum neutralizing-antibody titres, but not with cellular immune responses as measured by gamma interferon secretion by mouse splenocytes. Titres of serum neutralizing antibodies induced by the Ad S/N vaccine administered intranasally or intramuscularly were significantly lower than those induced by the WKV vaccine. However, Ad S/N administered intranasally, but not intramuscularly, significantly limited SARS-CoV replication in the lungs. Among the vaccine groups, SARS-CoV-specific IgA was found only in the sera of mice immunized intranasally with Ad S/N, suggesting that mucosal immunity may play a role in protection for the intranasal Ad S/N delivery system. Finally, the sera of vaccinated mice contained antibodies to S, further suggesting a role for this protein in conferring protective immunity against SARS-CoV infection.
Subject(s)
Antibodies, Viral/blood , Severe Acute Respiratory Syndrome/immunology , Severe Acute Respiratory Syndrome/prevention & control , Severe acute respiratory syndrome-related coronavirus/immunology , Vaccination , Viral Vaccines/administration & dosage , Administration, Intranasal , Animals , Antibodies, Viral/immunology , Antibody Specificity , Disease Models, Animal , Drug Evaluation, Preclinical , Female , Immunoglobulin A/blood , Immunoglobulin A/immunology , Injections, Intramuscular , Injections, Subcutaneous , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Mice , Neutralization Tests , Nucleocapsid Proteins/genetics , Severe acute respiratory syndrome-related coronavirus/chemistry , Spike Glycoprotein, Coronavirus , Vaccines, DNA/administration & dosage , Vaccines, DNA/genetics , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunologyABSTRACT
Severe acute respiratory syndrome coronavirus (SARS-CoV) has been identified as the aetiological agent of SARS. Thus, vaccination against SARS-CoV may represent an effective approach towards controlling SARS. The nucleocapsid (N) protein is thought to play a role in induction of cell-mediated immunity to SARS-CoV and thus it is important to characterize this protein. In the present study, an E1/partially E3-deleted, replication-defective human adenovirus 5 (Ad5) vector (Ad5-N-V) expressing the SARS-CoV N protein was constructed. The N protein, expressed in vitro by Ad5-N-V, was of the expected molecular mass of 50 kDa and was phosphorylated. Vaccination of C57BL/6 mice with Ad5-N-V generated potent SARS-CoV-specific humoral and T cell-mediated immune responses. These results show that Ad5-N-V may potentially be used as a SARS-CoV vaccine.