galK-based suicide vector mediated allelic exchange in Mycobacterium abscessus

Abstract

Mycobacterium abscessus is a fast-growing environmental organism and an important emerging pathogen. It is highly resistant to many antibiotics and undergoes a smooth to rough colony morphology change that appears to be important for pathogenesis. Smooth environmental strains have a glycopeptidolipid (GPL) on the surface, while certain types of clinical strains are often rough and lack this GPL, due to mutations in biosynthetic genes or the mmpL4b transporter gene. We report here the development and evaluation of an allelic exchange system for unmarked alleles in M. abscessus ATCC19977, using a suicide vector bearing the E. coli galK gene and 2-deoxygalactose counterselection. We describe here two variant galK suicide vectors, and demonstrate their utility in constructing a variety of mutants with deletion alleles of the mmpL4b GPL transporter gene, the mbtH GPL biosynthesis gene, the known b-lactamase gene MAB_2875 and a putative b-lactamase gene, MAB_2833. We also show that a novel allele of the E. coli aacC4 gene, conferring apramycin resistance (aacC41), can be used as a selectable marker in M. abscessus ATCC19977 at single copy.

In Silico Screen and Structural Analysis Identifies Bacterial Kinase Inhibitors which Act with β-Lactams To Inhibit Mycobacterial Growth

“…Mycobacterium abscessus PM3386 Δbla 61 ( Mab ) MIC determinations were done according to the microdilution method. Antibiotics were tested in two-fold serial dilutions with GSK690693 at 50 μM or with a DMSO control in a 96-well format.…”

Glycopeptidolipids, a Double-Edged Sword of the Mycobacterium abscessus Complex

“…Mycobacterium abscessus is a fast-growing non-tuberculous mycobacterium (NTM) and an emerging human pathogen that causes nosocomial skin and soft tissue infections ( Brown-Elliott et al, 2012 ) but also pulmonary infections, especially in patients with cystic fibrosis (CF) and other lung disorders ( Sermet-Gaudelus et al, 2003 ; Esther et al, 2010 ). Recent investigations reported mechanisms of virulence and physiopathological processes characterizing M. abscessus infection thanks to (i) genetic tools that allowed generation of defined mutants and transposon libraries, particularly useful to seek out genetic determinants of intracellular survival ( Medjahed and Reyrat, 2009 ; Cortes et al, 2011 ; Gregoire et al, 2017 ; Laencina et al, 2018 ) and (ii) the development of various complementary cellular and animal models, which have allowed delineation of the early stages of the infection and the role of important cell types participating in controlling the infection and/or in the formation of granulomas ( Ordway et al, 2008 ; Bernut et al, 2014a , 2017 ; Laencina et al, 2018 ). Evidence exists that granulomas harbor persistent M. abscessus for extended periods of time ( Tomashefski et al, 1996 ; Medjahed et al, 2010 ).…”

A Simple and Rapid Gene Disruption Strategy in Mycobacterium abscessus: On the Design and Application of Glycopeptidolipid Mutants

“…Information provided by such studies would likely bring answers to questions such as why M. abscessus is so resistant to such a large variety of drugs and why, despite being an environmental mycobacterium, it is capable of causing serious infections in humans. Indeed, recent years have seen the development of genetic tools to inactivate M. abscessus genes in a site-specific manner (Medjahed and Reyrat, 2009 ; Halloum et al, 2016 ; Gregoire et al, 2017 ; Rominski et al, 2017 ) as well as to generate conditional gene expression mutants (Cortes et al, 2011 ). This has led to the identification of a yet small number of genes that are downright essential for virulence or intracellular survival (Bernut et al, 2014 , 2016 ; Halloum et al, 2016 ) or playing more important roles during certain stages of infection, such as the establishment of infection (Bakala N'Goma et al, 2015 ) or during the chronic stage of infection (Viljoen et al, 2016 ).…”