Mutations in the β-Subunit of the RNA Polymerase Impair the Surface-Associated Motility and Virulence of Acinetobacter baumannii

Abstract

Acinetobacter baumannii is a major cause of antibiotic-resistant nosocomial infections worldwide. In this study, several rifampin-resistant spontaneous mutants obtained from the A. baumannii ATCC 17978 strain that differed in their point mutations in the rpoB gene, encoding the ␤-subunit of the RNA polymerase, were isolated. All the mutants harboring amino acid substitutions in position 522 or 540 of the RpoB protein were impaired in surface-associated motility and had attenuated virulence in the fertility model of Caenorhabditis elegans. The transcriptional profile of these mutants included six downregulated genes encoding proteins homologous to transporters and metabolic enzymes widespread among A. baumannii clinical isolates. The construction of knockout mutants in each of the six downregulated genes revealed a significant reduction in the surface-associated motility and virulence of four of them in the A. baumannii ATCC 17978 strain, as well as in the virulent clinical isolate MAR002. Taken together, our results provide strong evidence of the connection between motility and virulence in this multiresistant nosocomial pathogen.KEYWORDS Acinetobacter baumannii, surface-associated motility, rpoB, rifampin resistance A cinetobacter baumannii is a Gram-negative bacterium often responsible for nosocomial infections, based on its capacity to acquire and develop antimicrobial resistance (1). It is one of the most frequently encountered pathogens in intensive care, neonatal, and burn units, where it causes pneumonia and infections involving the central nervous system, skin, soft tissues, and bone (1). However, despite the increasing clinical importance of A. baumannii, little is known about the virulence factors that contribute to its pathogenetic properties.In previous studies of DNA damage-mediated mutagenesis in A. baumannii (2-5), we observed an atypical motility pattern in several spontaneous rifampin-resistant (Rif r ) mutants derived from the wild-type (WT) strain ATCC 17978 growing on semisolid medium. Although Acinetobacter spp. lack flagella, they exhibit surface-associated motility (6, 7). In a recent study, hypermotile A. baumannii derivatives with increasing virulence were isolated, but whether a lack of motility negatively affects the virulence and pathogenesis of A. baumannii is unclear (8, 9).The point mutations conferring rifampin resistance are located in the rpoB gene (encoding the ␤-subunit of the RNA polymerase), and they produce substantial changes in the transcriptional profile of bacterial cells by affecting several promoters (3, 10, 11). Nonetheless, not all mutations conferring rifampin resistance give rise to global modifications in the transcriptional profile of the respective bacterium (12, 13). In this RESULTSMotility and virulence of A. baumannii rpoB mutants. By plating saturated cultures of A. baumannii ATCC 17978 in the presence of rifampin (50 mg/liter), we isolated 10 spontaneous mutants (Rif r 1 to Rif r 10) resistant to the antimicrobial. In subsequent motility assays, ...

The mismatch repair system (mutS and mutL) in Acinetobacter baylyi ADP1

“…rpoB mutation in Clostridium difficile was associated with fitness cost in vitro and reduced virulence in vivo [21]. Mutations at position 522 or 540 in rpoB of A. baumannii were impaired in surface-associated motility and showed attenuated virulence [22]. The four different rpoB mutations in E. coli exhibited deleterious fitness costs under nutrient-rich conditions, but some rpoB alleles showed a remarkable fitness increase under phosphate limitation conditions [23].…”

Development of attenuated Vibrio parahaemolyticus mutant strains as potential live vaccines

“…There were 27 codons changed in the rpoB gene of the L4650 strain. The number of mutations in the rpoB gene was higher than that in some previous reports (Hazen et al, 2009;Perez-Varela et al, 2017;Qazi et al, 2014). However, the bacterial strains in those reports were cultured in medium containing Rif with concentrations 40, 50 and 100 μg/ml, while L4650 strains were grown over many generations in medium containing Rif with gradually increasing concentrations (from 10 to 250 μg/ml).…”

“…The full-length of the rpoB gene was 4029 bp encoding a polypeptide with 1342 amino acids (Campbell et al, 2001). Mutations in the rpoB gene were found to be associated with Rif resistance of some bacterial strains such as Mycobacterium tuberculosis, Escherichia coli, Acinetobacter baumannii, Bacillus subtilis, V. parahaemolyticus (Hazen et al, 2009;Jin and Gross, 1988;Maughan et al, 2004;Perez-Varela et al, 2017;Zaw et al, 2018), and the mutation position would affect the level of the resistance to Rif (Taniguchi et al, 1996). Hazen et al (2009) reported that V. parahaemolyticus Rif-resistant strains with concentration at 100 µg/ml had the mutations in the rpoB gene (Hazen et al, 2009).…”

“…The spontaneous resistance to Rif is often accompanied by the variations that change the fundamental structures of some proteins, however, the exact mechanism of attenuation of virulence in bacteria has not been clarified (Hu et al, 2012). Perez-Varela M. et al (2017) suggested that mutations in the βsubunit of RNA polymerase impaired the surfaceassociated motility and virulence of Acinetobacter baumannii (Perez-Varela et al, 2017).…”

A response regulator protein with antar domain, AvnR, in Acinetobacter baumannii ATCC 17978 impacts its virulence and amino acid metabolism

“…For example, expression of a predicted operon for a taurine ABC transporter, consisting of four genes (A1S_1442-44), was downregulated in avnR deletion mutant. A1S_1443 that encodes for a predicted taurine ATP-binding transport system component has previously been associated with an involvement of surfaceassociated motility and virulence of A. baumannii [58] where the deletion of A1S_1443 abolished motility and was less virulent in a C. elegans model. Further, taurine is also believed to protect bacterial biofilms from osmotic stress [59].…”