A Commensal Bacterium Promotes Virulence of an Opportunistic Pathogen via Cross-Respiration

Abstract

Bacteria rarely inhabit infection sites alone, instead residing in diverse, multispecies communities. Despite this fact, bacterial pathogenesis studies primarily focus on monoculture infections, overlooking how community interactions influence the course of disease. In this study, we used global mutant fitness profiling (transposon sequencing [Tn-seq]) to determine the genetic requirements for the pathogenic bacterium Aggregatibacter actinomycetemcomitans to cause disease when coinfecting with the commensal bacterium Streptococcus gordonii Our results show that S. gordonii extensively alters A. actinomycetemcomitans requirements for virulence factors and biosynthetic pathways during infection. In addition, we discovered that the presence of S. gordonii enhances the bioavailability of oxygen during infection, allowing A. actinomycetemcomitans to shift from a primarily fermentative to a respiratory metabolism that enhances its growth yields and persistence. Mechanistically, respiratory metabolism enhances the fitness of A. actinomycetemcomitans in vivo by increasing ATP yields via central metabolism and creating a proton motive force. Our results reveal that, similar to cross-feeding, where one species provides another species with a nutrient, commensal bacteria can also provide electron acceptors that promote the respiratory growth and fitness of pathogens in vivo, an interaction that we term cross-respiration.

Importance: Commensal bacteria can enhance the virulence of pathogens in mixed-species infections. However, knowledge of the mechanisms underlying this clinically relevant phenomenon is lacking. To bridge this gap, we comprehensively determined the genes a pathogen needs to establish coinfection with a commensal. Our findings show that the metabolism of the pathogen is low-energy-yielding in monoinfection, but in coinfection, the commensal improves the fitness of the pathogen by increasing the bioavailability of oxygen, thereby shifting the pathogen toward a high-energy-yielding metabolism. Similar to cross-feeding, this interaction, which we term cross-respiration, illustrates that commensal bacteria can provide electron acceptors that enhance the virulence of pathogens during infection.

FIG 1

Metabolic pathways required for anoxic and oxic growth in vitro. (A) Blue and orange indicate pathways required for anoxic and oxic growth, respectively. Each arrow represents an enzyme(s). The box embedded in the thick gray bar (symbolizing the membrane) represents the electron transport chain (ETC). The structure labeled ATP represents ATP synthase. TMAO, trimethylamine N-oxide; TMA, trimethylamine; DMSO, dimethyl sulfoxide; DMS, dimethyl sulfide; CoA, coenzyme A. (B) Cellular processes required for anoxic and oxic growth. See Tables S1 and S2 in Dataset S1 in the supplemental material for a full summary.

FIG 2

Anoxic growth is important for A. actinomycetemcomitans monoinfection. (A) Venn diagrams showing the overlap between monoinfection and anoxic (left) or oxic (right) growth in vitro. The percentages represent the percentage of anoxic (left) or oxic (right) fitness determinants that overlap monoinfection fitness determinants. The P values represent the significance of the enrichment of anoxic (left) or oxic (right) fitness determinants (one-tailed Fisher exact test). (B) Spearman’s rank correlation (r) between P values for the enrichment of COGs among monoinfection and anoxic (left) or oxic (right) fitness determinants (one-tailed Fisher exact test). Each point corresponds to an individual COG. (C) Biosynthetic requirements for monoinfection determined as described in the materials and methods. Metabolites in blue and orange are required for anoxic and oxic growth, respectively. PABA, p-aminobenzoic acid; SAM, S-adenosylmethionine; PLP, pyridoxal phosphate. See Table S5 in Dataset S1 in the supplemental material for a full summary. (D) Central metabolic pathways required for monoinfection. Green and red indicate pathways required for growth in the abscess and growth in vitro, respectively. Abbreviations not defined here are defined in the legend to Fig. 1 or in the text.

FIG 3

Coinfection stimulates A. actinomycetemcomitans oxic growth. (A) Venn diagrams showing the overlap between coinfection and anoxic (left) or oxic (right) growth in vitro. The percentages represent the percentage of anoxic (left) or oxic (right) fitness determinants that overlap coinfection fitness determinants. The P values represent the significance of the enrichment of anoxic (left) or oxic (right) fitness determinants (one-tailed Fisher exact test). (B) Spearman’s rank correlation (r) between P values for the enrichment of COGs among coinfection and anoxic (left) or oxic (right) fitness determinants (one-tailed Fisher exact test). Each point corresponds to an individual COG.

FIG 4

Fitness determinants specific to mono- and coinfection. (A) Venn diagram showing the numbers of fitness determinants specific to mono- and coinfection. Blue and orange indicate the percentages of fitness determinants associated with anoxic and oxic growth in vitro, respectively. (B) Cellular processes specific to mono- and coinfection. Processes in blue and orange are required for anoxic and oxic growth, respectively. cAMP, cyclic AMP. See Table S8 in Dataset S1 in the supplemental material for a full summary. (C) Spearman’s rank correlation (r) between P values for the enrichment of COGs among anoxic and monoinfection (left) or coinfection (right) fitness determinants (one-tailed Fisher exact test). Each point corresponds to an individual COG. (D) Central metabolic pathways required for mono- and coinfection. Green and purple indicate pathways required for mono- and coinfection, respectively. Abbreviations not defined here are defined in the legend to Fig. 1 or in the text.

FIG 5

S. gordonii exhibits synergy with wild-type (wt) A. actinomycetemcomitans and the A. actinomycetemcomitans ΔatpB mutant. Abscesses formed with the indicated strains were harvested at 3 days postinfection, and CFU were determined. Each symbol represents a single abscess. Data represent the results for 2 biological replicates (n ≥ 10 mice). % survival/abscess (y axis) was calculated using the output and input CFU/abscess. Statistical significance was determined by a two-tailed Mann-Whitney U test.