Biochemical and immunological characterization of a cpn60.1 knockout mutant of Mycobacterium bovis BCG.
Pathogenic mycobacteria possess two homologous chaperones encoded by cpn60.1 and cpn60.2. Cpn60.2 is essential for survival, providing the basic chaperone function, while Cpn60.1 is not. In the present study, we show that inactivation of the Mycobacterium bovis BCG cpn60.1 (Mb3451c) gene does not significantly affect bacterial growth in 7H9 broth, but that this knockout mutant (Δcpn60.1) forms smaller colonies on solid 7H11 medium than the parental and complemented strains. When growing on Sauton medium, the Δcpn60.1 mutant exhibits a thinner surface pellicle and is associated with higher culture filtrate protein content and, coincidentally, with less protein in its outermost cell envelope in comparison with the parental and complemented strains. Interestingly, in this culture condition, the Δcpn60.1 mutant is devoid of phthiocerol dimycocerosates, and its mycolates are two carbon atoms longer than those of the wild-type, a phenotype that is fully reversed by complementation. In addition, Δcpn60.1 bacteria are more sensitive to stress induced by H(2)O(2) but not by SDS, high temperature or acidic pH. Taken together, these data indicate that the cell wall of the Δcpn60.1 mutant is impaired. Analysis by 2D gel electrophoresis and MS reveals the upregulation of a few proteins such as FadA2 and isocitrate lyase in the cell extract of the mutant, whereas more profound differences are found in the composition of the mycobacterial culture filtrate, e.g. the well-known Hsp65 chaperonin Cpn60.2 is particularly abundant and increases about 200-fold in the filtrate of the Δcpn60.1 mutant. In mice, the Δcpn60.1 mutant is less persistent in lungs and, to a lesser extent, in spleen, but it induces a comparable mycobacteria-specific gamma interferon production and protection against Mycobacterium tuberculosis H37Rv challenge as do the parental and complemented BCG strains. Thus, by inactivating the cpn60.1 gene in M. bovis BCG we show that Cpn60.1 is necessary for the integrity of the bacterial cell wall, is involved in resistance to H(2)O(2)-induced stress but is not essential for its vaccine potential.
Scientific Institute of Public Health, Operational Direction of Communicable and Infectious Diseases, Rue Engeland 642, B-1180 Brussels, Belgium.
Xiao-Ming
XM
Wang
Scientific Institute of Public Health, Operational Direction of Communicable and Infectious Diseases, Rue Engeland 642, B-1180 Brussels, Belgium.
Changlong
C
Lu
Scientific Institute of Public Health, Operational Direction of Communicable and Infectious Diseases, Rue Engeland 642, B-1180 Brussels, Belgium.
Karine
K
Soetaert
Department of Proteomics and Microbiology, University of Mons, 20, place du Parc, B-7000 Mons, Belgium.
Catherine
C
S'Heeren
Scientific Institute of Public Health, Operational Direction of Communicable and Infectious Diseases, Rue Engeland 642, B-1180 Brussels, Belgium.
Priska
P
Peirs
Department of Molecular Mechanisms of the Mycobacterial Infections, Institute of Pharmacology and Structural Biology of CNRS and the University Paul Sabatier (UMR 5089), 205 route de Narbonne, Toulouse 31077 cedex 04, France.
Marie-Antoinette
MA
Lanéelle
Scientific Institute of Public Health, Operational Direction of Communicable and Infectious Diseases, Rue Engeland 642, B-1180 Brussels, Belgium.
Philippe
P
Lefèvre
Scientific Institute of Public Health, Operational Direction of Communicable and Infectious Diseases, Rue Engeland 642, B-1180 Brussels, Belgium.
Pablo
P
Bifani
Scientific Institute of Public Health, Operational Direction of Communicable and Infectious Diseases, Rue Engeland 642, B-1180 Brussels, Belgium.
Jean
J
Content
Department of Molecular Mechanisms of the Mycobacterial Infections, Institute of Pharmacology and Structural Biology of CNRS and the University Paul Sabatier (UMR 5089), 205 route de Narbonne, Toulouse 31077 cedex 04, France.
Mamadou
M
Daffé
Scientific Institute of Public Health, Operational Direction of Communicable and Infectious Diseases, Rue Engeland 642, B-1180 Brussels, Belgium.
Kris
K
Huygen
Scientific Institute of Public Health, Operational Direction of Communicable and Infectious Diseases, Rue Engeland 642, B-1180 Brussels, Belgium.
Jacqueline
J
De Bruyn
Department of Proteomics and Microbiology, University of Mons, 20, place du Parc, B-7000 Mons, Belgium.
Ruddy
R
Wattiez
D000818:Animals; D000900:Anti-Bacterial Agents / Q000633:toxicity; D058491:Bacterial Load; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D002473:Cell Wall / Q000737:chemistry / Q000502:physiology; D003470:Culture Media / Q000737:chemistry; D004195:Disease Models, Animal; D015180:Electrophoresis, Gel, Two-Dimensional; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D006861:Hydrogen Peroxide / Q000633:toxicity; D008055:Lipids / Q000737:chemistry; D008168:Lung / Q000382:microbiology; D013058:Mass Spectrometry; D051379:Mice; D008807:Mice, Inbred BALB C; D018832:Molecular Chaperones / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D009163:Mycobacterium bovis / Q000235:genetics / Q000254:growth & development / Q000276:immunology* / Q000502:physiology*; D009171:Mycolic Acids / Q000737:chemistry / Q000378:metabolism; D018384:Oxidative Stress; D020543:Proteome / Q000032:analysis; D012376:Rodent Diseases / Q000382:microbiology / Q000473:pathology; D012967:Sodium Dodecyl Sulfate / Q000633:toxicity; D013154:Spleen / Q000382:microbiology; D014376:Tuberculosis / Q000382:microbiology / Q000473:pathology
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D003470:Culture Media; D008055:Lipids; D018832:Molecular Chaperones; D009171:Mycolic Acids; D020543:Proteome; D012967:Sodium Dodecyl Sulfate; C008901:phthiocerol dimycocerosate; D006861:Hydrogen Peroxide