Simultaneous Identification of Potential Pathogenicity Factors of Mycoplasma agalactiae in the Natural Ovine Host by Negative Selection.
Mycoplasmas possess complex pathogenicity determinants that are largely unknown at the molecular level. Mycoplasma agalactiae serves as a useful model to study the molecular basis of mycoplasma pathogenicity. The generation and in vivo screening of a transposon mutant library of M. agalactiae were employed to unravel its host colonization factors. Tn4001mod mutants were sequenced using a novel sequencing method, and functionally heterogeneous pools containing 15 to 19 selected mutants were screened simultaneously through two successive cycles of sheep intramammary infections. A PCR-based negative selection method was employed to identify mutants that failed to colonize the udders and draining lymph nodes in the animals. A total of 14 different mutants found to be absent from ≥ 95% of samples were identified and subsequently verified via a second round of stringent confirmatory screening where 100% absence was considered attenuation. Using this criterion, seven mutants with insertions in genes MAG1050, MAG2540, MAG3390, uhpT, eutD, adhT, and MAG4460 were not recovered from any of the infected animals. Among the attenuated mutants, many contain disruptions in hypothetical genes, implying their previously unknown role in M. agalactiae pathogenicity. These data indicate the putative role of functionally different genes, including hypothetical ones, in the pathogenesis of M. agalactiae. Defining the precise functions of the identified genes is anticipated to increase our understanding of M. agalactiae infections and to develop successful intervention strategies against it.
Division of Clinical Microbiology and Infection Biology, Institute of Bacteriology, Mycology and Hygiene, Department of Pathobiology, University of Veterinary Medicine, Vienna, Austria.
Shivanand
S
Hegde
Division of Clinical Microbiology and Infection Biology, Institute of Bacteriology, Mycology and Hygiene, Department of Pathobiology, University of Veterinary Medicine, Vienna, Austria.
Shrilakshmi
S
Hegde
Division of Clinical Microbiology and Infection Biology, Institute of Bacteriology, Mycology and Hygiene, Department of Pathobiology, University of Veterinary Medicine, Vienna, Austria.
Martina
M
Zimmermann
Clinic for Ruminants, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria.
Martina
M
Flöck
Division of Clinical Microbiology and Infection Biology, Institute of Bacteriology, Mycology and Hygiene, Department of Pathobiology, University of Veterinary Medicine, Vienna, Austria.
Joachim
J
Spergser
Division of Clinical Microbiology and Infection Biology, Institute of Bacteriology, Mycology and Hygiene, Department of Pathobiology, University of Veterinary Medicine, Vienna, Austria.
Renate
R
Rosengarten
Division of Clinical Microbiology and Infection Biology, Institute of Bacteriology, Mycology and Hygiene, Department of Pathobiology, University of Veterinary Medicine, Vienna, Austria Rohini.Chopra-Dewasthaly@vetmeduni.ac.at.
Rohini
R
Chopra-Dewasthaly
D000818:Animals; D004251:DNA Transposable Elements; D055786:Gene Knockout Techniques; D005820:Genetic Testing; D008198:Lymph Nodes / Q000382:microbiology; D008321:Mammary Glands, Animal / Q000382:microbiology; D016254:Mutagenesis, Insertional; D009175:Mycoplasma Infections / Q000382:microbiology / Q000662:veterinary*; D045644:Mycoplasma agalactiae / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity*; D012756:Sheep; D012757:Sheep Diseases / Q000382:microbiology*; D037521:Virulence Factors / Q000235:genetics*
D004251:DNA Transposable Elements; D037521:Virulence Factors