Genome-Wide Identification of Acinetobacter baumannii Genes Necessary for Persistence in the Lung

Abstract

Acinetobacter baumannii is a Gram-negative bacterium that causes diseases such as pneumonia, bacteremia, and soft tissue infections in hospitalized patients. Relatively little is known about how A. baumannii causes these infections. Thus, we used insertion sequencing (INSeq), a combination of transposon mutagenesis and massively parallel next-generation sequencing, to identify novel virulence factors of A. baumannii. To this end, we generated a random transposon mutant library containing 150,000 unique insertions in A. baumannii strain ATCC 17978. The INSeq analysis identified 453 genes required for growth in rich medium. The library was then used in a murine pneumonia model, and the relative levels of abundance of mutants before and after selection in the mouse were compared. When genes required for growth in rich medium were removed from the analysis, 157 genes were identified as necessary for persistence in the mouse lung. Several of these encode known virulence factors of A. baumannii, such as OmpA and ZnuB, which validated our approach. A large number of the genes identified were predicted to be involved in amino acid and nucleotide metabolism and transport. Other genes were predicted to encode an integration host factor, a transmembrane lipoprotein, and proteins involved in stress response and efflux pumps. Very few genes, when disrupted, resulted in an increase in A. baumannii numbers during host infection. The INSeq approach identified a number of novel virulence determinants of A. baumannii, which are candidate targets for therapeutic interventions.IMPORTANCE A. baumannii has emerged as a frequent cause of serious infections in hospitals and community settings. Due to increasing antibiotic resistance, alternative approaches, such as antivirulence strategies, are desperately needed to fight A. baumannii infections. Thorough knowledge of A. baumannii pathogenicity is essential for such approaches but is currently lacking. With the increasingly widespread use of massively parallel sequencing, a class of techniques known as transposon insertion sequencing has been developed to perform comprehensive virulence screens of bacterial genomes in vivo. We have applied one of these approaches (INSeq) to uncover novel virulence factors in A. baumannii. We identified several such factors, including those predicted to encode amino acid and nucleotide metabolism proteins, an integration host factor protein, stress response factors, and efflux pumps. These results greatly expand the number of A. baumannii virulence factors and uncover potential targets for antivirulence treatments.

Genome-wide screen identifies host colonization determinants in a bacterial gut symbiont

“…Direct experimental approaches are not feasible for human gut symbionts, but rodent models have been used with considerable success (2-7). Several pathogens have been examined using Tn-seq in vivo, within different tissue types (38,(41)(42)(43). Some critical mechanisms for S. alvi colonization of the bee gut, including LPS modifications, membrane integrity components, the stringent response, proteases, and DNA break repair, are also important for infection by human pathogens (41)(42)(43).…”

“…Several pathogens have been examined using Tn-seq in vivo, within different tissue types (38,(41)(42)(43). Some critical mechanisms for S. alvi colonization of the bee gut, including LPS modifications, membrane integrity components, the stringent response, proteases, and DNA break repair, are also important for infection by human pathogens (41)(42)(43). Similarly, colonization of light-producing organs by the squid symbiont Vibrio fisheri depends on genes dictating biofilm formation and cellular stress responses (44).…”

Pathogenic Acinetobacter: from the Cell Surface to Infinity and Beyond

“…How A. baumannii partitions a given carbohydrate repeat unit to the protein glycosylation pathway or capsule production remains to be determined. Although the carbohydrate structures produced by different strains are highly variable, functional studies have shown that capsule production is essential for Acinetobacter survival during infection and growth in serum (32,46,50,51). It was recently reported that capsule production could be increased by the presence of subinhibitory levels of antibiotics, which increased resistance to complement-mediated killing and led to a hypervirulent phenotype in a mouse model of systemic infection (52).…”

“…Collectively, these findings indicate that the Acinetobacter T2SS is a previously unrecognized virulence factor mediating pathogenesis in a relevant mammalian model. Interestingly, a recent study by Wang et al utilized an A. baumannii ATCC 17978 gspN mutant for validation of their insertion sequencing murine pulmonary infection studies, and they subsequently found that the gspN mutant did not display any virulence defect in survival or competition models, compared to the parent strain (51). Although these data are in contrast to the newly defined role of type II secretion in Acinetobacter, it was demonstrated previously that gspN homologs were not required for a functioning T2SS in Klebsiella oxytoca (73); furthermore, gspN homologs are absent from numerous known T2SSs in other Gram-negative bacteria (74), indicating the dispensable nature of GspN in functioning T2SSs.…”

Resources for Genetic and Genomic Analysis of Emerging Pathogen Acinetobacter baumannii

“…baumannii research progress has been limited by the technologies and resources available for studying the pathogen. Although methods for targeted gene disruption, transposon mutagenesis, single-copy complementation using Tn7, and plasmid complementation have been developed (25)(26)(27)(28)(29)(30)(31), only a few resources have been described for genome-scale experimental studies (31). To facilitate large-scale genetic analysis of A. baumannii, we report here a set of genetic and genomic resources based on a …”