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. 2019 Nov 5;5:33.
doi: 10.1038/s41522-019-0105-6. eCollection 2019.

Reprioritization of Biofilm Metabolism Is Associated With Nutrient Adaptation and Long-Term Survival of Haemophilus influenzae

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Free PMC article

Reprioritization of Biofilm Metabolism Is Associated With Nutrient Adaptation and Long-Term Survival of Haemophilus influenzae

Alistair Harrison et al. NPJ Biofilms Microbiomes. .
Free PMC article

Abstract

Nontypeable Haemophilus influenzae (NTHI) is a human-restricted pathogen with an essential requirement for heme-iron acquisition. We previously demonstrated that microevolution of NTHI promotes stationary phase survival in response to transient heme-iron restriction. In this study, we examine the metabolic contributions to biofilm formation using this evolved NTHI strain, RM33. Quantitative analyses identified 29 proteins, 55 transcripts, and 31 metabolites that significantly changed within in vitro biofilms formed by RM33. The synthesis of all enzymes within the tryptophan and glycogen pathways was significantly increased in biofilms formed by RM33 compared with the parental strain. In addition, increases were observed in metabolite transport, adhesin production, and DNA metabolism. Furthermore, we observed pyruvate as a pivotal point in the metabolic pathways associated with changes in cAMP phosphodiesterase activity during biofilm formation. Taken together, changes in central metabolism combined with increased stores of nutrients may serve to counterbalance nutrient sequestration.

Keywords: Bacteriology; Biofilms.

Conflict of interest statement

Competing interestsThe authors declare no competing interests.

Figures

Fig. 1
Fig. 1
A persistent isolate of NTHI exhibits increased biofilm height and transformation efficiency. a Schematic representation of environmental heme–iron restriction. 86-028NP was cultured in defined iron source (DIS) medium in the presence (+) or absence (−) of 2 µg/mL heme–iron for 24 h to generate “replete” or “restricted” populations, respectively, adapted from Hardison et al. These two populations were subcultured into DIS medium containing 2 µg/mL heme–iron. The “continuously exposed” or “transiently restricted” cultures were continuously incubated under static microaeration with no fresh medium added. Viability was determined by plating for CFU/mL every 24 h, each bar indicates the CFU on that day for a representative experiment. The persistent isolate, RM33, was isolated from the transiently restricted culture on day 33 (arrow). b Representative image of a biofilm of 86-028NP (pGM1.1) grown for 48 h with orthogonal views of the three-dimensional rendering of a 20.4 µm high biofilm. c Representative image of a biofilm of RM33 (pKM1.1) grown for 48 h with orthogonal views of the three-dimensional rendering of a 38.4 µm high biofilm. d Representative image of a 13.8 µm biofilm with a 1:1 mixture of 86-028 NP (pGM1.1) and RM33 (pKM1.1) grown for 48 h with orthogonal views of the three-dimensional rendering. e Quantitative assessment of the height of biofilms formed by either the parental strain [86-028NP (pGM1.1), circle] and [RM33 (pKM1.1), square]. The height of the biofilm was measured at ten random locations in technical duplicate and performed on six independent occasions (n = 120 per strain). Each point represents an individual measurement. Statistical significance was determined by a two-tailed unpaired Student’s t test. f Quantitative assessment of the height of each strain within a mixed biofilm was measured as described in e. g The phosphodiesterase activity of whole-cell lysates prepared from 48 h biofilms was determined as described in “Methods” section. The mean and standard error of the mean (s.e.m.) are indicated for the results of five independent assays. Statistical significance was determined using a two-tailed unpaired Student’s t test. h Exogenous DNA was added to a 48 h biofilm of either 86-028NP or RM33. Biofilms were physically disrupted and the efficiency of DNA uptake is reported as the number of antibiotic-resistant colonies within the total population. The mean and standard error of the mean (s.e.m.) are indicated for the results of four independent assays. Statistical significance was determined using a two-tailed unpaired Student’s t test. Scale bar indicates 25 μm
Fig. 2
Fig. 2
Amino acid metabolism and entry into the TCA cycle during biofilm growth is increased in RM33 biofilms. The pathways of amino acids that are associated with the TCA cycle were determined from the information provided for 86-028NP in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Biocyc Pathway databases. The concentration of amino acids within the biofilms of 86-028NP (circle) and RM33 (square) were quantitated as described in “Methods” section. The red X indicates absence of known orthologs of enzymes involved in the TCA cycle for 86-028NP. The enzymes encircled in purple are significantly increased in RM33 biofilms. The enzyme encircled in yellow is significantly decreased in RM33 biofilms. The concentrations of aspartate (pink cloud) and serine (green cloud) in the spent media were below the level of detection (indicated by the white highlight). pp periplasm, cyto cytoplasm. The mean and s.e.m. are depicted for each metabolite from three independent biological biofilms grown for 48 h. Statistical significance was determined using a two-tailed unpaired Student’s t test (*p < 0.036; **p = 0.004)
Fig. 3
Fig. 3
Metabolites associated with the urea cycle are significantly increased in RM33 biofilms. The concentrations of the metabolites associated with the urea cycle determined from information at KEGG and Biocyc were quantified as described in “Methods” section. The red X indicates absence of known orthologs of enzymes involved in the urea cycle for 86-028NP. The mean and s.e.m. of the concentrations for the metabolites from three independent biological replicates of 86-028NP (circle) and RM33 (square) 48 h biofilms are presented. Statistical significance was determined using a two-tailed unpaired Student’s t test (*p < 0.025)
Fig. 4
Fig. 4
Production of polyamines differs between 86-028NP and RM33 biofilms. The classical pathways for polyamine synthesis were adapted from KEGG. The red X indicates absence of known orthologs of enzymes involved in polyamine synthesis for 86-028NP. The mean and s.e.m. of the concentrations of the metabolites within three independent biological replicates 86-028NP (circle) and RM33 (square) 48 h biofilms were quantified as described in “Methods” section. Statistical significance was determined using a two-tailed unpaired Student’s t test (*p < 0.02; **p = 0.005). pp periplasm, cyto cytoplasm
Fig. 5
Fig. 5
Tryptophan biosynthesis enzymes are significantly increased in RM33 biofilms. a The enzymatic pathway for synthesis of tryptophan was adapted from KEGG. The concentration of amino acids within the biofilms of 86-028NP (circle) and RM33 (square) and secreted indole were quantitated from three independent biological replicates as described in the “Methods” section. The TnaB importer of tryptophan from the periplasm (pp) to the cytoplasm (cyto) was not significantly different between the two strains. Statistical significance was determined using a two-tailed unpaired Student’s t test (*p = 0.03; **p = 0.007; ***p < 0.0001). b Representative image of a biofilm of the parent (86-028NP) grown for 48 h and stained with LIVE/DEAD to visualize DNA stain with orthogonal views of the three-dimensional rendering of a 26.4 µm biofilm. c Representative image of a biofilm of 86-028NP trpBA mutant grown for 48 h and stained with LIVE/DEAD to visualize bacteria with orthogonal views of the three-dimensional rendering of a 6 µm biofilm. d Representative image of a biofilm of 86-028NP trpBA tnaAB mutant grown for 48 h and stained with LIVE/DEAD to visualize bacteria with orthogonal views of the three-dimensional rendering of a 6 µm biofilm. e Quantitative assessment of the height of biofilms formed by the parental strain (86-028 NP), 86-028NP trpBA, or 86-028NP trpBA tnaAB. The height of the biofilm was measured at ten random locations on three independent occasions (n = 30 per strain). Each point represents an individual measurement. Statistical significance was determined by a two-tailed unpaired Student’s t test. f The culture supernatant from three biofilms grown for 48 h on three independent occasions were used to quantify the level of indole as described in the “Methods” section. The dashed horizontal line indicates the limit of detection. The mean and s.e.m. are reported. Statistical significance was determined using a two-tailed unpaired Student’s t test. Scale bar indicates 25 μm

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