Copy Number of an Integron-Encoded Antibiotic Resistance Locus Regulates a Virulence and Opacity Switch in Acinetobacter baumannii AB5075

Abstract

We describe a novel genetic mechanism in which tandem amplification of a plasmid-borne integron regulates virulence, opacity variation, and global gene expression by altering levels of a putative small RNA (sRNA) in Acinetobacter baumannii AB5075. Copy number of this amplified locus correlated with the rate of switching between virulent opaque (VIR-O) and avirulent translucent (AV-T) cells. We found that prototypical VIR-O colonies, which exhibit high levels of switching and visible sectoring with AV-T cells by 24 h of growth, harbor two copies of this locus. However, a subset of opaque colonies that did not form AV-T sectors within 24 h were found to harbor only one copy. The colonies with decreased sectoring to AV-T were designated low-switching opaque (LSO) variants and were found to exhibit a 3-log decrease in switching relative to that of the VIR-O. Overexpression studies revealed that the element regulating switching was localized to the 5′ end of the aadB gene within the amplified locus. Northern blotting indicated that an sRNA of approximately 300 nucleotides (nt) is encoded in this region and is likely responsible for regulating switching to AV-T. Copy number of the ∼300-nt sRNA was also found to affect virulence, as the LSO variant exhibited decreased virulence during murine lung infections. Global transcriptional profiling revealed that >100 genes were differentially expressed between VIR-O and LSO variants, suggesting that the ∼300-nt sRNA may act as a global regulator. Several virulence genes exhibited decreased expression in LSO cells, potentially explaining their decreased virulence. IMPORTANCE Acinetobacter baumannii remains a leading cause of hospital-acquired infections. Widespread multidrug resistance in this species has prompted the WHO to name carbapenem-resistant A. baumannii as its top priority for research and development of new antibiotics. Many strains of A. baumannii undergo a high-frequency virulence switch, which is an attractive target for new therapeutics targeting this pathogen. This study reports a novel mechanism controlling the frequency of switching in strain AB5075. The rate of switching from the virulent opaque (VIR-O) to the avirulent translucent (AV-T) variant is positively influenced by the copy number of an antibiotic resistance locus encoded on a plasmid-borne composite integron. Our data suggest that this locus encodes a small RNA that regulates opacity switching. Low-switching opaque variants, which harbor a single copy of this locus, also exhibit decreased virulence. This study increases our understanding of this critical phenotypic switch, while also identifying potential targets for virulence-based A. baumannii treatments.

Integron Functionality and Genome Innovation: An Update on the Subtle and Smart Strategy of Integrase and Gene Cassette Expression Regulation

“…Several studies have proposed that the aminoglycoside-inducible translation of aac and aad genes inserted in class 1 integrons involved the participation of regulatory RNAs, such as riboswitches and small RNAs (SRNAs) (for revision, see references [130][131][132][133][134]). However, it was verified that the DNA sequence coding for the proposed riboswitch RNA actually corresponded to the attI1 site of class 1 integrons.…”

“…In bacteria, sRNAs act as global regulators of mRNAs, modulating their activity at the posttranscriptional level. A recent study characterized a new mechanism in which in tandem amplification of integron-associated gene cassettes would alter the levels of a putative small RNA (sRNA), which would regulate virulence and global gene expression in A. baumannii [134]. These transcripts can be derived from non-coding sequences, such as the UTRs of genes and intergenic regions, and can be independently expressed or produced by the cleavage of longer transcripts catalyzed by RNAses [136].…”

“…Some A. baumannii strains may switch between the virulent phenotype of opacity (VIR-O) and the avirulent translucent (AV-T), resulting in decreased virulence in vivo [137]. Anderson et al [134] observed that a duplication of an aadB-containing gene cassette array in a composite class 1 integron positively influenced the rates of switching between VIR-O and AV-T cells. However, they noticed that another bacterial subpopulation displaying a third opacity phenotype, designated low-switching opaque (LSO) variant, exhibited a remarkable reduction of switching to AV-T relative to the switching frequency of the VIR-O variants.…”

Flagellum and toxin phase variation impacts intestinal colonization and disease development in a mouse model of Clostridioides difficile infection

“…1,3 In many pathogens including Acinetobacter baumannii, Bordetella bronchiseptica, and Streptococcus pneumoniae, host selective pressures can substantially affect the composition of a phase-variable bacterial population during infection. [4][5][6][7][8][9][10][11] All three of these pathogens can form distinct subpopulations differentially equipped for survival in disparate environments. However, determining the importance of phase variation (rather than the phase-variable trait) in pathogenesis can be challenging.…”

Presentation_1.pptx

“…Both variants switch back and forth at high frequency, with rates of approximately 4-13% conversion at 24 hours of colony growth and 20-40% at 48 hours (Chin et al, 2018). The two types have distinctly different genomic expression profiles as revealed by RNA sequencing, and a variety of gene productsincluding transcriptional regulators, a two-component system (OmpR/EnvZ), an efflux pump (ArpB), and a putative sRNA located upstream of a plasmid-encoded antibiotic resistance locus-appear to contribute to interconversion between VIR-O and AV-T (Tipton and Rather, 2016;Tipton et al, 2017;Chin et al, 2018;Anderson et al, 2020). One such regulator, ABUW_1645 (1645), is a TetR-type transcriptional regulator (TTTR) whose rate of expression is 150-fold higher in the AV-T state and whose overexpression in the VIR-O background drives conversion to the AV-T state (Chin et al, 2018).…”