Comunicación oral en Reunión del Grupo de Microbiología Molecular de la SEM

6 de septiembre de 2016

Hoefer A., Gutierrez B., Martinez-Ovejero C., Delgado-Blas JF., Santos-Lopez A., Thomas-Lopez D. y Gonzalez-Zorn B.

In the last decade an increasing number of 16S rRNA methyltransferases conferring unusually high-level pan-resistance to aminoglycosides have been rapidly disseminating amongst clinically relevant Gram-negative pathogens. Phenotypic characteristics coupled with in silico modelling have identified a putative regulatory domain in the 5’ UTR of armA, the most predominant 16S methyltransferase worldwide. By performing rapid amplification of cDNA ends (RACE) we found that the full 5’ UTR of the armA transcript is 139 nucleotides. This region along with its corresponding promoter is 100% conserved in all genetic environments of arma identified to date. Phenotypic changes upon truncation of the 5’ UTR, such as MIC reduction, demonstrate the involvement of the upstream region in the expression of the resistance determinant. To further elucidate the role of the armA upstream domain, we constructed 5’ UTR:lacZα reporter constructs. The results indicated a constitutive expression of ArmA both in the presence and absence of aminoglycosides. We then designed and performed a qRT-PCR, which also showed a constitutive expression of armA, suggesting that regulation could be dependent on a post-transcriptional mechanism. To investigate the translational frequency of active ArmA we then tagged the resistance methylase with a FlagTag in its C-terminus. The Westren Blot results revealed the differential expression of the truncations, which directly correlate to the phenotypic changes previously observed. To further characterise methylase expression throughout the life-cycle of the cell, we performed comprehensive Western blot growth curves. Under the control of the full-length 5’ UTR there is not only a significant increase in the overall expression of armA but also a significant initial peak early in the exponential phase. Our results are the first to highlight the importance of posttranscriptional regulation of acquired 16S rRNA methylases, where the presence of the active enzyme correlates with the number of ribosomes to be methylated. This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement N° 289285.

	Servicio de Zoonosis de Transmisión Alimentaria y Resistencia a Antimicrobianos (ZTA). Centro de Vigilancia Sanitaria Veterinaria (VISAVET). Universidad Complutense (UCM).
	Departamento de Sanidad Animal. Facultad de Veterinaria. Universidad Complutense (UCM).

Enlace a Reunión del Grupo de Microbiología Molecular de la SEM