The central role of arginine in Haemophilus influenzae survival in a polymicrobial environment with Streptococcus pneumoniae and Moraxella catarrhalis

Abstract

Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis are bacterial species which frequently co-colonise the nasopharynx, but can also transit to the middle ear to cause otitis media. Chronic otitis media is often associated with a polymicrobial infection by these bacteria. However, despite being present in polymicrobial infections, the molecular interactions between these bacterial species remain poorly understood. We have previously reported competitive interactions driven by pH and growth phase between H. influenzae and S. pneumoniae. In this study, we have revealed competitive interactions between the three otopathogens, which resulted in reduction of H. influenzae viability in co-culture with S. pneumoniae and in triple-species culture. Transcriptomic analysis by mRNA sequencing identified a central role of arginine in mediating these interactions. Arginine supplementation was able to increase H. influenzae survival in a dual-species environment with S. pneumoniae, and in a triple-species environment. Arginine was used by H. influenzae for ATP production, and levels of ATP generated in dual- and triple-species co-culture at early stages of growth were significantly higher than the combined ATP levels of single-species cultures. These results indicate a central role for arginine-mediated ATP production by H. influenzae in the polymicrobial community.

Fig 1. Growth of H. influenzae in dual- and triple-species co-cultures with S. pneumoniae and M. catarrhalis after 18 h.

Growth in (A) planktonic and (B) biofilm state of H. influenzae middle ear isolate 86-028NP and laboratory strain Rd KW20 in mono-culture, in co-culture with S. pneumoniae strain 11, in co-culture with M. catarrhalis strain QC, and in triple-species co-culture with S. pneumoniae 11 and M. catarrhalis QC after 18h is represented as CFU/mL. * p<0.05, *** p<0.001.

Fig 2. Growth dynamics of dual- and triple-species co-cultures of H. influenzae, S. pneumoniae and M. catarrhalis after 2 h.

Growth in mono-, dual- and triple-species co-culture of (A), H. influenzae 86-028NP, (B), S. pneumoniae 11, and (C), M. catarrhalis QC in the planktonic state after 2h of growth are demonstrated as CFU/mL.

Fig 3. Arginine restores H. influenzae survival in dual- and triple-species co-culture.

Viable cell numbers of H. influenzae Rd KW20 or H. influenzae 86-028NP in planktonic (A,B) and biofilm (C,D) growth in mono-culture, dual-culture with S. pneumoniae 11, dual-culture with M. catarrhalis QC, and in triple-species culture for 18h, with and without supplementation with exogenous arginine at a concentration of 4g/L. Dotted line indicates the limit of detection. *, p<0.05.

Fig 4. Arginine supplementation enhances H. influenzae ATP production.

Relative ATP production measured by luminescence (relative light units, RLU), of H. influenzae without supplementation of arginine (black), and with supplementation of 2 g/L (black dotted line), 4 g/L arginine (grey filled line) and 6 g/L arginine (grey dotted line). Luminescence (ATP production) is significantly higher in all concentrations of arginine, compared to no arginine supplementation. Significant differences were determined with a student t-test (*** p ≤ 0.001, ** p ≤ 0.01).

Fig 5. ATP production is enhanced in dual- and triple-species cultures.

ATP production measured by luminescence (relative light units, RLU), of H. influenzae 86-028NP (86), S. pneumoniae 11 and M. catarrhalis QC following 2 h of incubation in single-species culture, dual-species culture, and triple species culture at A) pH 8, with and without arginine supplementation, and B) in pH 7, without arginine supplementation. Significant differences were observed between H. influenzae alone (86), in dual-species culture with S. pneumoniae (86+11), and in triple-species culture (Triple). Statistical significance was evaluated with a student’s t-test (** p<0.01, *** p<0.001, **** p≤0.0001).

Fig 6. Arginine import and catabolic pathways in H. influenzae 86-028NP as determined from KEGG pathway analysis.

Pathways for which genes have been identified in 86-028NP are indicated with black line, and pathways for which genes are missing in 86-028NP are indicated with dashed line.