Analysis of Six tonB Gene Homologs in Bacteroides fragilis Revealed That tonB3 is Essential for Survival in Experimental Intestinal Colonization and Intra-Abdominal Infection

Abstract

The opportunistic, anaerobic pathogen and commensal of the human large intestinal tract, Bacteroides fragilis strain 638R, contains six predicted TonB proteins, termed TonB1-6, four ExbBs orthologs, ExbB1-4, and five ExbDs orthologs, ExbD1-5. The inner membrane TonB/ExbB/ExbD complex harvests energy from the proton motive force (Δp) and the TonB C-terminal domain interacts with and transduces energy to outer membrane TonB-dependent transporters (TBDTs). However, TonB’s role in activating nearly one hundred TBDTs for nutrient acquisition in B. fragilis during intestinal colonization and extraintestinal infection has not been established. In this study, we show that growth was abolished in the ΔtonB3 mutant when heme, vitamin B12, Fe(III)-ferrichrome, starch, mucin-glycans, or N-linked glycans were used as a substrate for growth in vitro . Genetic complementation of the ΔtonB3 mutant with the tonB3 gene restored growth on these substrates. The ΔtonB1 , ΔtonB2 , ΔtonB4, ΔtonB5, and ΔtonB6 single mutants did not show a growth defect. This indicates that there was no functional compensation for the lack of TonB3, and it demonstrates that TonB3, alone, drives the TBDTs involved in the transport of essential nutrients. The ΔtonB3 mutant had a severe growth defect in a mouse model of intestinal colonization compared to the parent strain. This intestinal growth defect was enhanced in the ΔtonB3 ΔtonB6 double mutant strain which completely lost its ability to colonize the mouse intestinal tract compared to the parent strain. The ΔtonB1 , ΔtonB2 , ΔtonB4, and ΔtonB5 mutants did not significantly affect intestinal colonization. Moreover, the survival of the ΔtonB3 mutant strain was completely eradicated in a rat model of intra-abdominal infection. Taken together, these findings show that TonB3 was essential for survival in vivo . The genetic organization of tonB1 , tonB2 , tonB4, tonB5, and tonB6 gene orthologs indicates that they may interact with periplasmic and nonreceptor outer membrane proteins, but the physiological relevance of this has not been defined. Because anaerobic fermentation metabolism yields a lower Δp than aerobic respiration and B. fragilis has a reduced redox state in its periplasmic space - in contrast to an oxidative environment in aerobes - it remains to be determined if the diverse system of TonB/ExbB/ExbD orthologs encoded by B. fragilis have an increased sensitivity to PMF (relative to aerobic bacteria) to allow for the harvesting of energy under anaerobic conditions.

The dual role of TonB genes in turnerbactin uptake and carbohydrate utilization in the shipworm symbiontTeredinibacter turnerae

“…The TonB system was originally found and has been extensively characterized in E. coli (Postle and Larsen, 2007). E. coli and many other bacteria carry a single set of the TonB system, but after finding two TonB systems in V. cholerae (Occhino et al, 1998), multiple TonB systems have been identified and characterized in a number of bacteria, including many Vibrio species (2 or 3 systems) (Seliger et al, 2001;Stork et al, 2004;Alice et al, 2008;Wang et al, 2008;Tanabe et al, 2012), Aeromonas hydrophila (3 systems) (Dong et al, 2016;Dong et al, 2019;Dong et al, 2023), Pseudomonas aeruginosa (3 systems) (Poole et al, 1996;Zhao and Poole, 2000;Huang et al, 2004), Acinetobacter baumannii (3 systems) (Zimbler et al, 2013;Runci et al, 2019), and Bacteroides fragilis (6 systems) (Parker et al, 2022). In those examples, some TonB systems are functionally independent while others show functionally redundancy, for transport for particular substances such as siderophores and other nutrients, or physiological activities.…”

The dual role of TonB genes in turnerbactin uptake and carbohydrate utilization in the shipworm symbiont Teredinibacter turnerae

“…The TonB system was originally found and has been extensively characterized in E. coli ( 65 ). E. coli and many other bacteria carry a single set of the TonB system, but after finding two TonB systems in V. cholerae ( 50 ), multiple TonB systems have been identified and characterized in a number of bacteria, including many Vibrio species (two or three systems) ( 51 , 52 , 66 – 68 ), Aeromonas hydrophila (three systems) ( 69 – 71 ), Pseudomonas aeruginosa (three systems) ( 63 , 72 , 73 ), Acinetobacter baumannii (three systems) ( 74 , 75 ), and Bacteroides fragilis (six systems) ( 76 ). In those examples, some TonB systems are functionally independent while others show functional redundancy, for transport for particular substances such as siderophores and other nutrients, or physiological activities.…”

Roles of SmeYZ, SbiAB, and SmeDEF Efflux Systems in Iron Homeostasis of Stenotrophomonas maltophilia

“…The TonB/ExbB/ExbD system is required to energize several TonB dependent outer membrane transporters (TBDTs), which play a role in transportation of serval nutrients such as ferri-siderophores, vitamin B12, heme, metals and oligosaccharides from outside environments ( 29 – 31 ). A bacterium generally harbors multiple TonB genes, whose encoded proteins seem to specifically interact with subsets of TBDTs for the transport of different substrates responsible for diverse physiological activities ( 32 – 35 ). There are five annotated TonB genes, Smlt0009, Smlt2939, Smlt3094, Smlt3477, and Smlt3892, in S. maltophilia genome.…”