Mycoplasma bovis 5'-nucleotidase, rather than membrane-associated nuclease MnuA, plays a pivotal role in pathogen survival, fitness, and virulence in bovine mastitis

Authors: GELGIE, AGA - University Of Tennessee; SCHNEIDER, PELEG - Hebrew University Of Jerusalem; CITTI, CHRISTINE - University Of Toulouse; DORDET-FRISONI, EMILIE - University Of Toulouse; GILLESPIE, BARBARA - University Of Tennessee; ALMEIDA, RAUL - University Of Tennessee; Agga, Getahun; AMOAH, YAA - Hebrew University Of Jerusalem; SHPIGEL, NAHUM - Hebrew University Of Jerusalem; LYSNYANSKY, INNA - Kimron Veterinary Institute; KERRO DEGO, OUDESSA - University Of Tennessee

Submitted to: PLoS Pathogens
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/30/2024
Publication Date: N/A
Citation: N/A

Interpretive Summary: Mycoplasma bovis is one of the major contagious mastitis causing bacterial species in dairy cattle. In addition to causing pain and inflammation to the udders of cows and overall wellbeing, mastitis reduces milk production. Beyond animal welfare, mastitis causes severe economic loss to dairy cattle farmers due to lost production and discarding of milk from affected cows. Mastitis has a broad societal impact by affecting the entire milk production industry, processing, distribution, and consumers. Coupled with farm hygiene practices, the use of antibiotics to prevent mastitis from occurring and treat clinically sick cows. Unfortunately, the use of antibiotics for these purposes could not be sustained to bacteria developing resistance to the commonly used antibiotics in U.S. dairy cattle production. There is also consumer pressure towards no antibiotic use in food animal production. To overcome these issues, alternative to antibiotics such as vaccines are urgently needed for food animal production. This study developed two mutant strains of a bacterial species of Mycoplasma and characterized them for infectivity and pathogenecity in causing mammary gland infections in cows. The study particularly identified a mutant strain that has the potential to be used as vaccine candidate. Further research is needed to further explore the bacterial variant. If proved to be effective, the commercialization of this product will increase the economic benefit of dairy cattle production.

Technical Abstract: Nucleases as well as 5' nucleotidases (5'-NT) play an essential role in central pathways of cell biology and are often associated with bacterial virulence. In Mycoplasma spp., which have limited metabolic capacities and rely on nutrient availability, these enzymes are of significant importance. This study explores the potential role of the major membrane nuclease MnuA and 5'-NT in Mycoplasma bovis-associated mastitis. Mutants in MBOVPG45_0215 (mnuA) and MBOVPG45_0690 (5'-NT) were identified through genome-wide transposon mutagenesis and their impact on the fitness and virulence of M. bovis PG45 type strain was validated both in vitro in axenic medium and in vivo using murine and cow mastitis models. Disruption of mnuA resulted in decreased nuclease activity, while the '0690 mutant exhibited slow log-phase growth, reduced colony size, and impaired hydrolase activity towards ATP, 5' and 3'AMPs, thereby combining 5'-/3'-nucleosidase and nucleoside triphosphate diphosphohydrolase activities. Using murine and cow mastitis models, we compared the disease elicited by M. bovis wild type and its '0215 and '0690 isogenic mutants. Inactivation of MBOVPG45_0690 reduced M. bovis fitness in mammary glands, as evidenced by a significant decrease or absence in post-challenge bacterial counts in murine and cow mammary tissues, respectively. Moreover, mammary inflammation, measured by the relative expression of major inflammatory genes (in murine model) or by somatic cell counts (SCC), inflammatory changes in milk and udder tissue or by ability to recruit neutrophils (in cow model), were significantly reduced only with '0690 mutant. In contrast, the '0215 mutant activated inflammatory genes, caused an increase in SCC, recruit neutrophils, and induce subclinical mastitis in cows in a manner comparable to the WT. Collectively, the assessment of fitness and pathogenesis of '0215 and '0690 mutants, using both murine and cow mastitis models, yielded complementary insights and mutually reinforced results revealing that disruption of the 5'-NT leads to a significant attenuation of M. bovis, whereas mycoplasma with disrupted mnuA gene still causes mastitis.