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Experimental infections with Mycoplasma agalactiae identify key factors involved in host-colonization.

Baranowski E, Bergonier D, Sagné E, Hygonenq MC, Ronsin P, Berthelot X, Citti C - PLoS ONE (2014)

Bottom Line: In these PG2-infected ewes, we observed over the course of infection (i) the development of a specific antibody response and (ii) dynamic changes in expression of M. agalactiae surface variable proteins (Vpma), with multiple Vpma profiles co-existing in the same animal.In contrast and despite a sensitive model, none of the knock-out mutants were able to survive and colonize the host.The extreme avirulent phenotype of the two mutants was further supported by the absence of an IgG response in inoculated animals.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR 1225, IHAP, Toulouse, France; Université de Toulouse, INP-ENVT, UMR 1225, IHAP, Toulouse, France.

ABSTRACT
Mechanisms underlying pathogenic processes in mycoplasma infections are poorly understood, mainly because of limited sequence similarities with classical, bacterial virulence factors. Recently, large-scale transposon mutagenesis in the ruminant pathogen Mycoplasma agalactiae identified the NIF locus, including nifS and nifU, as essential for mycoplasma growth in cell culture, while dispensable in axenic media. To evaluate the importance of this locus in vivo, the infectivity of two knock-out mutants was tested upon experimental infection in the natural host. In this model, the parental PG2 strain was able to establish a systemic infection in lactating ewes, colonizing various body sites such as lymph nodes and the mammary gland, even when inoculated at low doses. In these PG2-infected ewes, we observed over the course of infection (i) the development of a specific antibody response and (ii) dynamic changes in expression of M. agalactiae surface variable proteins (Vpma), with multiple Vpma profiles co-existing in the same animal. In contrast and despite a sensitive model, none of the knock-out mutants were able to survive and colonize the host. The extreme avirulent phenotype of the two mutants was further supported by the absence of an IgG response in inoculated animals. The exact role of the NIF locus remains to be elucidated but these data demonstrate that it plays a key role in the infectious process of M. agalactiae and most likely of other pathogenic mycoplasma species as many carry closely related homologs.

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Genetic and antigenic features of M. agalactiae strain PG2 and NifS mutants used for animal inoculation.Knock-out mutants NifS1 and NifS2 were generated by transposon mutagenesis in M. agalactiae reference strain PG2 [18]. (A) Drawing illustrating the NIF locus (grey arrows) in M. agalactiae. Genes are represented according to their orientations on the genome. Transposon insertion sites in NifS mutants are indicated by a vertical bar with a closed circle, an arrow indicate its orientation. The insertion of the transposon in mutant NifS2 occurred within the stop codon of nifS[18]. The scale is indicated. (B) SDS-PAGE analysis of Triton X-114 soluble materials extracted from cultures of PG2 and NifS mutants followed by Coomassie staining or immunodetection using the PAL97 hyperimmune serum. Molecular weight standards (MW) are in kDa. (C) Characterization of phase variable, Vpma expression profile in M. agalactiae inoculums. Vpma lipoproteins U, V, W, X, Y and Z were detected using specific polyclonal antibodies (see Materials and Methods). A lipoprotein P80 antiserum was used as control. (D) Growth curves of PG2 and NifS mutants in SP4-HS medium. Mycoplasma titers used for inoculations were 107 CFU/ml (left panel) and 105 CFU/ml (right panel). The data are the means of three independent assays. Standard deviations are indicated by error bars.
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pone-0093970-g001: Genetic and antigenic features of M. agalactiae strain PG2 and NifS mutants used for animal inoculation.Knock-out mutants NifS1 and NifS2 were generated by transposon mutagenesis in M. agalactiae reference strain PG2 [18]. (A) Drawing illustrating the NIF locus (grey arrows) in M. agalactiae. Genes are represented according to their orientations on the genome. Transposon insertion sites in NifS mutants are indicated by a vertical bar with a closed circle, an arrow indicate its orientation. The insertion of the transposon in mutant NifS2 occurred within the stop codon of nifS[18]. The scale is indicated. (B) SDS-PAGE analysis of Triton X-114 soluble materials extracted from cultures of PG2 and NifS mutants followed by Coomassie staining or immunodetection using the PAL97 hyperimmune serum. Molecular weight standards (MW) are in kDa. (C) Characterization of phase variable, Vpma expression profile in M. agalactiae inoculums. Vpma lipoproteins U, V, W, X, Y and Z were detected using specific polyclonal antibodies (see Materials and Methods). A lipoprotein P80 antiserum was used as control. (D) Growth curves of PG2 and NifS mutants in SP4-HS medium. Mycoplasma titers used for inoculations were 107 CFU/ml (left panel) and 105 CFU/ml (right panel). The data are the means of three independent assays. Standard deviations are indicated by error bars.

Mentions: M. agalactiae reference strain PG2 was used in this study [10]. Mutants NifS1 and NifS2, originally described as 7.82 and 7.134, were selected from a library of transposon knock-out mutants generated in strain PG2 [18]. They both carry a transposon in a genomic region designated as the NIF locus (CDS MAG0720 to MAG0730) (Fig. 1). The reference strain PG2 and the NifS mutants were cultured at 37°C in a modified SP4 medium, termed as SP4-HS, in which horse serum (HS) was substituted for fetal bovine serum. The SP4-HS medium was supplemented with 500 μg/ml cefalexin (Virbac). Mycoplasma cultures were stored at −80°C, and CFU titers were determined by serial dilutions in Dubelco’s phosphate-buffered saline (DPBS; Invitrogen) supplemented with 1% heat inactivated HS (Invitrogen). Dilutions were spotted (10 μl) onto solid SP4-HS medium and mycoplasma colonies were counted after 2 to 5 days incubation at 37°C. The absence of contaminating wild-type sequences in culture stocks of NifS mutants was tested by PCR amplification, as previously described [18].


Experimental infections with Mycoplasma agalactiae identify key factors involved in host-colonization.

Baranowski E, Bergonier D, Sagné E, Hygonenq MC, Ronsin P, Berthelot X, Citti C - PLoS ONE (2014)

Genetic and antigenic features of M. agalactiae strain PG2 and NifS mutants used for animal inoculation.Knock-out mutants NifS1 and NifS2 were generated by transposon mutagenesis in M. agalactiae reference strain PG2 [18]. (A) Drawing illustrating the NIF locus (grey arrows) in M. agalactiae. Genes are represented according to their orientations on the genome. Transposon insertion sites in NifS mutants are indicated by a vertical bar with a closed circle, an arrow indicate its orientation. The insertion of the transposon in mutant NifS2 occurred within the stop codon of nifS[18]. The scale is indicated. (B) SDS-PAGE analysis of Triton X-114 soluble materials extracted from cultures of PG2 and NifS mutants followed by Coomassie staining or immunodetection using the PAL97 hyperimmune serum. Molecular weight standards (MW) are in kDa. (C) Characterization of phase variable, Vpma expression profile in M. agalactiae inoculums. Vpma lipoproteins U, V, W, X, Y and Z were detected using specific polyclonal antibodies (see Materials and Methods). A lipoprotein P80 antiserum was used as control. (D) Growth curves of PG2 and NifS mutants in SP4-HS medium. Mycoplasma titers used for inoculations were 107 CFU/ml (left panel) and 105 CFU/ml (right panel). The data are the means of three independent assays. Standard deviations are indicated by error bars.
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Related In: Results  -  Collection

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pone-0093970-g001: Genetic and antigenic features of M. agalactiae strain PG2 and NifS mutants used for animal inoculation.Knock-out mutants NifS1 and NifS2 were generated by transposon mutagenesis in M. agalactiae reference strain PG2 [18]. (A) Drawing illustrating the NIF locus (grey arrows) in M. agalactiae. Genes are represented according to their orientations on the genome. Transposon insertion sites in NifS mutants are indicated by a vertical bar with a closed circle, an arrow indicate its orientation. The insertion of the transposon in mutant NifS2 occurred within the stop codon of nifS[18]. The scale is indicated. (B) SDS-PAGE analysis of Triton X-114 soluble materials extracted from cultures of PG2 and NifS mutants followed by Coomassie staining or immunodetection using the PAL97 hyperimmune serum. Molecular weight standards (MW) are in kDa. (C) Characterization of phase variable, Vpma expression profile in M. agalactiae inoculums. Vpma lipoproteins U, V, W, X, Y and Z were detected using specific polyclonal antibodies (see Materials and Methods). A lipoprotein P80 antiserum was used as control. (D) Growth curves of PG2 and NifS mutants in SP4-HS medium. Mycoplasma titers used for inoculations were 107 CFU/ml (left panel) and 105 CFU/ml (right panel). The data are the means of three independent assays. Standard deviations are indicated by error bars.
Mentions: M. agalactiae reference strain PG2 was used in this study [10]. Mutants NifS1 and NifS2, originally described as 7.82 and 7.134, were selected from a library of transposon knock-out mutants generated in strain PG2 [18]. They both carry a transposon in a genomic region designated as the NIF locus (CDS MAG0720 to MAG0730) (Fig. 1). The reference strain PG2 and the NifS mutants were cultured at 37°C in a modified SP4 medium, termed as SP4-HS, in which horse serum (HS) was substituted for fetal bovine serum. The SP4-HS medium was supplemented with 500 μg/ml cefalexin (Virbac). Mycoplasma cultures were stored at −80°C, and CFU titers were determined by serial dilutions in Dubelco’s phosphate-buffered saline (DPBS; Invitrogen) supplemented with 1% heat inactivated HS (Invitrogen). Dilutions were spotted (10 μl) onto solid SP4-HS medium and mycoplasma colonies were counted after 2 to 5 days incubation at 37°C. The absence of contaminating wild-type sequences in culture stocks of NifS mutants was tested by PCR amplification, as previously described [18].

Bottom Line: In these PG2-infected ewes, we observed over the course of infection (i) the development of a specific antibody response and (ii) dynamic changes in expression of M. agalactiae surface variable proteins (Vpma), with multiple Vpma profiles co-existing in the same animal.In contrast and despite a sensitive model, none of the knock-out mutants were able to survive and colonize the host.The extreme avirulent phenotype of the two mutants was further supported by the absence of an IgG response in inoculated animals.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR 1225, IHAP, Toulouse, France; Université de Toulouse, INP-ENVT, UMR 1225, IHAP, Toulouse, France.

ABSTRACT
Mechanisms underlying pathogenic processes in mycoplasma infections are poorly understood, mainly because of limited sequence similarities with classical, bacterial virulence factors. Recently, large-scale transposon mutagenesis in the ruminant pathogen Mycoplasma agalactiae identified the NIF locus, including nifS and nifU, as essential for mycoplasma growth in cell culture, while dispensable in axenic media. To evaluate the importance of this locus in vivo, the infectivity of two knock-out mutants was tested upon experimental infection in the natural host. In this model, the parental PG2 strain was able to establish a systemic infection in lactating ewes, colonizing various body sites such as lymph nodes and the mammary gland, even when inoculated at low doses. In these PG2-infected ewes, we observed over the course of infection (i) the development of a specific antibody response and (ii) dynamic changes in expression of M. agalactiae surface variable proteins (Vpma), with multiple Vpma profiles co-existing in the same animal. In contrast and despite a sensitive model, none of the knock-out mutants were able to survive and colonize the host. The extreme avirulent phenotype of the two mutants was further supported by the absence of an IgG response in inoculated animals. The exact role of the NIF locus remains to be elucidated but these data demonstrate that it plays a key role in the infectious process of M. agalactiae and most likely of other pathogenic mycoplasma species as many carry closely related homologs.

Show MeSH
Related in: MedlinePlus