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Novel Genes Required for Surface-Associated Motility in Acinetobacter baumannii.

ABSTRACT: Acinetobacter baumannii is an opportunistic and increasingly multi-drug resistant human pathogen rated as a critical priority one pathogen for the development of new antibiotics by the WHO in 2017. Despite the lack of flagella, A. baumannii can move along wet surfaces in two different ways: via twitching motility and surface-associated motility. While twitching motility is known to depend on type IV pili, the mechanism of surface-associated motility is poorly understood. In this study, we established a library of 30 A. baumannii ATCC® 17978™ mutants that displayed deficiency in surface-associated motility. By making use of natural competence, we also introduced these mutations into strain 29D2 to differentiate strain-specific versus species-specific effects of mutations. Mutated genes were associated with purine/pyrimidine/folate biosynthesis (e.g. purH, purF, purM, purE), alarmone/stress metabolism (e.g. Ap4A hydrolase), RNA modification/regulation (e.g. methionyl-tRNA synthetase), outer membrane proteins (e.g. ompA), and genes involved in natural competence (comEC). All tested mutants originally identified as motility-deficient in strain ATCC® 17978™ also displayed a motility-deficient phenotype in 29D2. By contrast, further comparative characterization of the mutant sets of both strains regarding pellicle biofilm formation, antibiotic resistance, and virulence in the Galleria mellonella infection model revealed numerous strain-specific mutant phenotypes. Our studies highlight the need for comparative analyses to characterize gene functions in A. baumannii and for further studies on the mechanisms underlying surface-associated motility.

SUBMITTER: Blaschke U

PROVIDER: S-EPMC7997844 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Acinetobacter baumannii is emerging as a leading global multiple-antibiotic-resistant nosocomial pathogen. The identity of genes essential for pathogenesis in a mammalian host remains largely unknown. Using transposon-directed insertion-site sequencing (TraDIS), we identified A. baumannii genes involved in bacterial survival in a leukopenic mouse model of bloodstream infection. Mice were inoculated with a pooled transposon mutant library derived from 109,000 mutants, and TraDIS was used to map transposon insertion sites in the genomes of bacteria in the inoculum and of bacteria recovered from mouse spleens. Unique transposon insertion sites were mapped and used to calculate a fitness factor for every insertion site based on its relative abundance in the inoculum and postinfection libraries. Eighty-nine transposon insertion mutants that were underrepresented after experimental infection in mice compared to their presence in the inocula were delineated as candidates for further evaluation. Genetically defined mutants lacking feoB (ferrous iron import), ddc (d-ala-d-ala-carboxypeptidase), and pntB (pyridine nucleotide transhydrogenase subunit) exhibited a fitness defect during systemic infection resulting from bacteremia. In vitro, these mutants, as well as a fepA (ferric enterobactin receptor) mutant, are defective in survival in human serum and within macrophages and are hypersensitive to killing by antimicrobial peptides compared to the survival of the parental strain under these conditions. Our data demonstrate that FepA is involved in the uptake of exogenous enterobactin in A. baumannii. Genetic complementation rescues the phenotypes of mutants in assays that emulate conditions encountered during infection. In summary, we have determined novel A. baumannii fitness genes involved in the pathogenesis of mammalian infection. IMPORTANCE A. baumannii is a significant cause of bacterial bloodstream infection in humans. Since multiple antibiotic resistance is becoming more common among strains of A. baumannii, there is an urgent need to develop novel tools to treat infections caused by this dangerous pathogen. To develop knowledge-guided treatment approaches for A. baumannii, a thorough understanding of the mechanism by which this pathogen causes bloodstream infection is required. Here, using a mouse model of infection, we report the identification of A. baumannii genes that are critical for the ability of this pathogen to cause bloodstream infections. This study lays the foundation for future research on A. baumannii genes that can be targeted to develop novel therapeutics against this emerging human pathogen.

Project description:UNLABELLED:Acinetobacter baumannii is a Gram-negative, opportunistic pathogen. Recently, multiple A. baumannii genomes have been sequenced; these data have led to the identification of many genes predicted to encode proteins required for the biogenesis of type IV pili (TFP). However, there is no experimental evidence demonstrating that A. baumannii strains actually produce functional TFP. Here, we demonstrated that A. baumannii strain M2 is naturally transformable and capable of twitching motility, two classical TFP-associated phenotypes. Strains were constructed with mutations in pilA, pilD, and pilT, genes whose products have been well characterized in other systems. These mutants were no longer naturally transformable and did not exhibit twitching motility. These TFP-associated phenotypes were restored when these mutations were complemented. More PilA was detected on the surface of the pilT mutant than the parental strain, and TFP were visualized on the pilT mutant by transmission electron microscopy. Thus, A. baumannii produces functional TFP and utilizes TFP for both natural transformation and twitching motility. Several investigators have hypothesized that TFP might be responsible, in part, for the flagellum-independent surface-associated motility exhibited by many A. baumannii clinical isolates. We demonstrated that surface-associated motility was not dependent on the products of the pilA, pilD, and pilT genes and, by correlation, TFP. The identification of functional TFP in A. baumannii lays the foundation for future work determining the role of TFP in models of virulence that partially recapitulate human disease. IMPORTANCE:Several investigators have documented the presence of genes predicted to encode proteins required for the biogenesis of TFP in many A. baumannii genomes. Furthermore, some have speculated that TFP may play a role in the unique surface-associated motility phenotype exhibited by many A. baumannii clinical isolates, yet there has been no experimental evidence to prove this. Unfortunately, progress in understanding the biology and virulence of A. baumannii has been slowed by the difficulty of constructing and complementing mutations in this species. Strain M2, a recently characterized clinical isolate, is amenable to genetic manipulation. We have established a reproducible system for the generation of marked and/or unmarked mutations using a modified recombineering strategy as well as a genetic complementation system utilizing a modified mini-Tn7 element in strain M2. Using this strategy, we demonstrated that strain M2 produces TFP and that TFP are not required for surface-associated motility exhibited by strain M2.

Project description:The important nosocomial pathogen Acinetobacter baumannii presents a quorum sensing (QS) system (abaI/abaR) mediated by acyl-homoserine-lactones (AHLs) and several quorum quenching (QQ) enzymes. However, the roles of this complex network in the control of the expression of important virulence-related phenotypes such as surface-associated motility and biofilm formation is not clear. Therefore, the effect of the mutation of the AHL synthase AbaI, and the exogenous addition of the QQ enzyme Aii20J on surface-associated motility and biofilm formation by A. baumannii ATCC® 17978TM was studied in detail. The effect of the enzyme on biofilm formation by several multidrug-resistant A. baumannii clinical isolates differing in their motility pattern was also tested. We provide evidence that a functional QS system is required for surface-associated motility and robust biofilm formation in A. baumannii ATCC® 17978TM. Important differences were found with the well-studied strain A. nosocomialis M2 regarding the relevance of the QS system depending on environmental conditions The in vitro biofilm-formation capacity of A. baumannii clinical strains was highly variable and was not related to the antibiotic resistance or surface-associated motility profiles. A high variability was also found in the sensitivity of the clinical strains to the action of the QQ enzyme, revealing important differences in virulence regulation between A. baumannii isolates and confirming that studies restricted to a single strain are not representative for the development of novel antimicrobial strategies. Extracellular DNA emerges as a key component of the extracellular matrix in A. baumannii biofilms since the combined action of the QQ enzyme Aii20J and DNase reduced biofilm formation in all tested strains. Results demonstrate that QQ strategies in combination with other enzymatic treatments such as DNase could represent an alternative approach for the prevention of A. baumannii colonization and survival on surfaces and the prevention and treatment of infections caused by this pathogen.

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Novel Genes Required for Surface-Associated Motility in Acinetobacter baumannii.

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