Metabolite cross-feeding enhances virulence in a model polymicrobial infection

Abstract

Microbes within polymicrobial infections often display synergistic interactions resulting in enhanced pathogenesis; however, the molecular mechanisms governing these interactions are not well understood. Development of model systems that allow detailed mechanistic studies of polymicrobial synergy is a critical step towards a comprehensive understanding of these infections in vivo. In this study, we used a model polymicrobial infection including the opportunistic pathogen Aggregatibacter actinomycetemcomitans and the commensal Streptococcus gordonii to examine the importance of metabolite cross-feeding for establishing co-culture infections. Our results reveal that co-culture with S. gordonii enhances the pathogenesis of A. actinomycetemcomitans in a murine abscess model of infection. Interestingly, the ability of A. actinomycetemcomitans to utilize L-lactate as an energy source is essential for these co-culture benefits. Surprisingly, inactivation of L-lactate catabolism had no impact on mono-culture growth in vitro and in vivo suggesting that A. actinomycetemcomitans L-lactate catabolism is only critical for establishing co-culture infections. These results demonstrate that metabolite cross-feeding is critical for A. actinomycetemcomitans to persist in a polymicrobial infection with S. gordonii supporting the idea that the metabolic properties of commensal bacteria alter the course of pathogenesis in polymicrobial communities.

Figure 1. Aerobic and anaerobic metabolites produced by A. actinomycetemcomitans, A. actinomycetemcomitans lctD ^- and A. actinomycetemcomitans cydB ^-.

Resting cell suspensions of each culture were incubated (A )aerobically in glucose; (B), anaerobically in glucose; (C), aerobically or anaerobically in lactate. Metabolite concentrations were measured by HPLC. Data in A and B is presented as moles of metabolite produced/mole of glucose consumed. Only trace concentrations (<50 µM) of ethanol were observed in anaerobic suspensions. Error bars represent 1 standard error of the mean, n = 3. * Acetate concentrations are shown per mM of L-lactate consumed. The detection limit for acetate was 100 µM.

Figure 2. Growth of A. actinomycetemcomitans, A. actinomycetemcomitans lctD ^-, and S. gordonii in aerobic and anaerobic co-cultures.

Strains were grown as mono- or co-cultures in 3 mM glucose aerobically or anaerobically for 10 or 12 h respectively, serially diluted and plated on selective media to determine colony forming units per ml (CFU/ml). A. actinomycetemcomitans mono-culture strains are black bars and co-culture with S. gordonii are white bars. Error bars represent 1 standard error of the mean, n = 3.

Figure 3. Metabolite production by A. actinomycetemcomitans, A. actinomycetemcomitans lctD ^-, and S. gordonii in aerobic or anaerobic co-cultures.

Supernatants of the cultures used for CFU measurements in Fig. 2 were analyzed by HPLC for metabolite production from (A), aerobic or (B), anaerobic cultures. Data is presented as moles of metabolite produced/mole of glucose consumed. Error bars represent 1 standard error of the mean, n = 3. ND  =  No Data.

Figure 4. Persistence of A. actinomycetemcomitans, A. actinomycetemcomitans lctD ^-, and A. actinomycetemcomitans apiA ^- in mono- or co-culture in a murine abscess model.

A. Bacterial colony forming units per abscess. Wilcoxon signed-rank test values are: * p<0.02, ** p<0.01, *** p<0.008. B. Abscess weights 6 days post-inoculation. Error bars represent 1 standard error of the mean, n = 9. p<0.05 for wt A. actinomycetemcomitans in mono- and co-culture via Student's t-test.

Figure 5. Model for electron transport during L-lactate oxidation in A. actinomycetemcomitans.

A. actinomycetemcomitans requires O₂ for oxidation of L-lactate. LctD may donate electrons from L-lactate directly to the quinone pool or utilize an unknown intermediate electron carrier represented by the dotted arrow. The cytochrome oxidase CydAD ultimately donates the electrons to O₂.

Figure 6. Model for enhanced persistence of A. actinomycetemcomitans during aerobic co-culture with S. gordonii.

During co-culture aerobic growth with glucose, S. gordonii produces L-lactate and H₂O₂ which inhibit A. actinomycetemcomitans glucose uptake (red line) and induce apiA expression (dotted line) respectively. The production of L-lactate provides A. actinomycetemcomitans with a preferred carbon source for growth and reduces the need to compete with S. gordonii for glucose during aerobic co-culture. During anaerobic co-culture, S. gordonii also produces L-lactate but A. actinomycetemcomitans is unable to catabolize this carbon source due to the absence of O₂; thus requiring A. actinomycetemcomitans to compete directly with S. gordonii for glucose (dashed line).