The Multidrug Efflux System AcrABZ-TolC Is Essential for Infection of Salmonella Typhimurium by the Flagellum-Dependent Bacteriophage Chi

Abstract

Bacteriophages are the most abundant biological entities in the biosphere. Due to their host specificity and ability to kill bacteria rapidly, bacteriophages have many potential healthcare applications, including therapy against antibiotic-resistant bacteria. Infection by flagellotropic bacteriophages requires a properly rotating bacterial flagellar filament. The flagella-dependent phage χ (Chi) infects serovars of the pathogenic enterobacterium Salmonella enterica. However, cell surface receptors and proteins involved in other stages of χ infection have not been discovered to date. We screened a multi-gene deletion library of S. enterica serovar Typhimurium by spotting mutants on soft agar plates seeded with bacteriophage χ and monitoring their ability to grow and form a swim ring, a characteristic of bacteriophage-resistant motile mutants. Those multi-gene deletion regions identified to be important for χ infectivity were further investigated by characterizing the phenotypes of corresponding single-gene deletion mutants. This way, we identified motile mutants with varying degrees of resistance to χ. Deletions in individual genes encoding the AcrABZ-TolC multi-drug efflux system drastically reduced infection by bacteriophage χ. Furthermore, an acrABtolC triple deletion strain was fully resistant to χ. Infection was severely reduced but not entirely blocked by the deletion of the gene tig encoding the molecular chaperone trigger factor. Finally, deletion in genes encoding enzymes involved in the synthesis of the antioxidants glutathione (GSH) and uric acid resulted in reduced infectivity. Our findings begin to elucidate poorly understood processes involved in later stages of flagellotropic bacteriophage infection and informs research aimed at the use of bacteriophages to combat antibiotic-resistant bacterial infections. IMPORTANCE Antimicrobial resistance is a large concern in the healthcare field. With more multi-drug resistant bacterial pathogens emerging, other techniques for eliminating bacterial infections are being explored. Among these is phage therapy, where combinations of specific phages are used to treat infections. Generally, phages utilize cell appendages and surface receptors for the initial attachment to their host. Phages that are flagellotropic are of particular interest because flagella are often important in bacterial virulence, making resistance to attachment of these phages harder to achieve without reducing virulence. This study discovered the importance of a multi-drug efflux pump for the infection of Salmonella enterica by a flagellotropic phage. In theory, if a bacterial pathogen develops phage resistance by altering expression of the efflux pump then the pathogen would simultaneously become more susceptible to the antibiotic substrates of the pump. Thus, co-administering antibiotics and flagellotropic phage may be a particularly potent antibacterial therapy.

Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria

“… 197 Similarly, in Salmonella Typhimurium, the TolC appears to be essential for the infection by the Chi-phage. 198 Likewise, TolC from V. cholerae is also utilized by the VP3 phage to gain entry into cells. 199 The AcrEF-TolC complex from E. coli has been posited to play a role in the maintenance of normal cell division.…”

Identification of Receptor Binding Proteins in Flagellotropic Agrobacterium Phage 7-7-1

“…Initial interactions are mediated by phage encoded RBPs. Flagellotropic phages first bind to the bacterial flagellar filament and have been shown to interact with secondary cell surface receptors such as LPS, efflux pumps, and CPS [ 13 , 14 , 40 , 47 ]. RBPs in non-flagellotropic phages have been identified, resulting in a wealth of knowledge surrounding common features of these proteins including resistance to proteolysis, thermostability, and the ability to degrade glycans [ 16 , 20 , 21 , 24 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 64 ].…”

“…However, phage-host interactions during viral entry are typically investigated unilaterally, largely focusing on the bacterial hosts. Such studies have identified outer membrane proteins, efflux pumps, capsular polysaccharide (CPS), lipopolysaccharide (LPS), pili, and flagella as receptors used by bacteriophages [ 8 , 9 , 10 , 11 , 12 , 13 , 14 ]. Inarguably, a holistic understanding of the mechanisms underlying phage entry entails equally detailed analyses of phage components involved in interacting with bacterial receptors.…”

MDR Pumps as Crossroads of Resistance: Antibiotics and Bacteriophages

“…The adsorption of bacteriophages to the surface of bacteria is usually described by several stages: (1) initial contact, (2) reversible binding, and (3) irreversible attachment [ 128 , 147 ]. Therefore, the question arises about the relationship between receptors (porins, pumps, channels) and the ability of bacteriophage nucleic acids to penetrate into cells [ 148 ]. Whether bacteriophages use the structural features of receptors for penetration through the outer membrane or only for recognition and attachment to the surface of bacteria remains debatable.…”