Mutation of the Carboxy-Terminal Processing Protease in Acinetobacter baumannii Affects Motility, Leads to Loss of Membrane Integrity, and Reduces Virulence

Abstract

Motility plays an essential role in the host–parasite relationship of pathogenic bacteria, and is often associated with virulence. While many pathogenic bacteria use flagella for locomotion, Acinetobacter baumannii strains do not have flagella, but have other features that aid in their motility. To study the genes involved in motility, transposon mutagenesis was performed to construct A. baumannii mutant strains. Mutant strain MR14 was found to have reduced motility, compared to wild-type ATCC 17978. NCBI BLAST analysis revealed that the Tn10 transposon in the MR14 genome is integrated into the gene that encodes for carboxy-terminal processing protease (Ctp). Additionally, MR14 exhibits a mucoidy, sticky phenotype as the result of increased extracellular DNA (eDNA) caused by bacterial autolysis. Transmission and scanning electron microscopy revealed cytoplasmic content leaving the cell and multiple cell membrane depressions, respectively. MR14 showed higher sensitivity to environmental stressors. Mutation of the ctp gene reduced invasion and adhesion of A. baumannii to airway epithelial cells, potentially due to increased hydrophobicity. In the zebrafish model of infection, MR14 increased the survival rate by 40% compared to the wild-type. Taken together, the ctp gene in A. baumannii has a pivotal role in maintaining membrane integrity, adaptation to environmental stress, and controlling virulence.

Acinetobacter baumannii Strains Deficient in the Clp Chaperone-Protease Genes Have Reduced Virulence in a Murine Model of Pneumonia

“…In addition to ClpA, ClpB and ClpS, the genome of A. baumannii AB5075 is predicted to encode several common protease-chaperones including ClpP, ClpU, Lon, Ctp and Prc [ 18 , 30 , 46 ]. While these factors have been well-described in other organisms, the function of these proteins, particularly during infection, are poorly understood in A. baumannii .…”

“…For example, Lon modulates biofilm formation and cell envelope integrity, though the specific mechanism through which Lon operates remains unclear [ 47 ]. Mutants lacking the C-terminal protease Ctp are attenuated in both cell culture and whole-animal infection assays [ 46 ]. Both, Lon and Ctp, are important in protecting A. baumannii against environmental stress, suggesting the importance of chaperone-proteases in pathogenesis or antibiotic resistance.…”

Recent Advances in Genetic Tools for Acinetobacter baumannii

“…Mariner ( Di Venanzio et al, 2019 ; Kazi et al, 2020 ; Lopez et al, 2020 ; Sun et al, 2020 ) and Tn 10 ( Giles et al, 2015 ; Geisinger et al, 2019 , 2020 ; Roy et al, 2020 ) transposons also prove useful, in A. baumannii . The suicide vector pJNW684 containing the himar1 mariner transposase, transposon and RP4/oriT/oriR6K origin of replication requires the λpir gene product for replication ( Wang et al, 2014 ).…”

“…A similar library in ATCC ® 17978 TM laid the foundation for the discovery that suppression of the T6SS allows for dissemination of MDR plasmids within A. baumannii but also within the Acinetobacter species itself ( Di Venanzio et al, 2019 ). Tn 10 delivery via either electroporation or conjugation, pBSL180 ( Roy et al, 2020 ) pDL1073 ( Geisinger et al, 2019 , 2020 ), and pLof ( Giles et al, 2015 ) have successfully generated libraries for A. baumannii. The use of pBSL180 ( Alexeyev and Shokolenko, 1995 ) has been marked in other Gram-negatives such as E. coli ( Scaletsky et al, 2005 ), Shenwanella oneidensis and Desulfovibrio desulfuricans ( Groh et al, 2005 ).…”

“…The use of pBSL180 ( Alexeyev and Shokolenko, 1995 ) has been marked in other Gram-negatives such as E. coli ( Scaletsky et al, 2005 ), Shenwanella oneidensis and Desulfovibrio desulfuricans ( Groh et al, 2005 ). In ATCC ® 17978 TM , the role of carboxy-terminal processing protease in motility and membrane integrity was characterized ( Roy et al, 2020 ). A drawback of using plasmid-based systems is the risk of integration of the backbone into the mutant chromosome.…”