Proteomic analysis of metronidazole resistance in the human facultative pathogen Bacteroides fragilis

Abstract

The anaerobic gut bacteria and opportunistic pathogen Bacteroides fragilis can cause life-threatening infections when leaving its niche and reaching body sites outside of the gut. The antimicrobial metronidazole is a mainstay in the treatment of anaerobic infections and also highly effective against Bacteroides spp. Although resistance rates have remained low in general, metronidazole resistance does occur in B. fragilis and can favor fatal disease outcomes. Most metronidazole-resistant Bacteroides isolates harbor nim genes, commonly believed to encode for nitroreductases which deactivate metronidazole. Recent research, however, suggests that the mode of resistance mediated by Nim proteins might be more complex than anticipated because they affect the cellular metabolism, e.g., by increasing the activity of pyruvate:ferredoxin oxidoreductase (PFOR). Moreover, although nim genes confer only low-level metronidazole resistance to Bacteroides, high-level resistance can be much easier induced in the laboratory in the presence of a nim gene than without. Due to these observations, we hypothesized that nim genes might induce changes in the B. fragilis proteome and performed comparative mass-spectrometric analyses with B. fragilis 638R, either with or without the nimA gene. Further, we compared protein expression profiles in both strains after induction of high-level metronidazole resistance. Interestingly, only few proteins were repeatedly found to be differentially expressed in strain 638R with the nimA gene, one of them being the flavodiiron protein FprA, an enzyme involved in oxygen scavenging. After induction of metronidazole resistance, a far higher number of proteins were found to be differentially expressed in 638R without nimA than in 638R with nimA. In the former, factors for the import of hemin were strongly downregulated, indicating impaired iron import, whereas in the latter, the observed changes were not only less numerous but also less specific. Both resistant strains, however, displayed a reduced capability of scavenging oxygen. Susceptibility to metronidazole could be widely restored in resistant 638R without nimA by supplementing growth media with ferrous iron sulfate, but not so in resistant 638R with the nimA gene. Finally, based on the results of this study, we present a novel hypothetic model of metronidazole resistance and NimA function.

Keywords: Bacteroides fragilis; antimicrobial resistance; metronidazole; nimA gene; proteomics.

Figure 1

Proteins identified to be differentially expressed in Bacteroides fragilis 638R^R as compared to 638R. (A) Total number of proteins differentially expressed as compared to wildtype 638R and their cellular localization. (B) Pie chart indicating the functions of the proteins differentially expressed in 638R^R.

Figure 2

Levels of fprA mRNA as determined by RT-qPCR in 638R, 638R^R, 638R nimA, 638R nimA^R, and 638R pFDfprA which carries an additional copy of the fprA gene on shuttle plasmid pFD340 under transcriptional control of insertion element 4351 (IS4351). Measurements were performed in three independent experiments each. The asterisk indicates 638R pFDfprA > 638R with p < 0.001 according to Brown-Forsythe and Welch ANOVA tests with a post-hoc two stage linear step-up procedure of Benjamini, Krieger, and Yekutieli.

Figure 3

Susceptibilities of (A) 638R and 638R^R, and (B) 638R nimA and 638R nimA^R as determined by Etest, either with or without supplementation of 100 μM ferrous iron sulfate. Due to the fainter appearance of 638R^R as compared to 638R, the point of intersection with the Etest strip is indicated by a black arrow.

Figure 4

Hypothetical scheme of the interdependence of Bacteroides fragilis metabolism, induced metronidazole resistance, and of NimA-mediated protection from metronidazole, respectively. The main metabolic end products are indicated in red, the enzymes involved in blue, ATP in green, and NADH in brown. In the left panel (i) the pathways in B. fragilis leading to the formation of the metabolic end products (Frantz and McCallum, 1979) are depicted. Glucose is broken down to phosphoenolpyruvate (PEP) which can be either converted to oxaloacetate by phosphoenolpyruvate carboxykinase (PEPCK) or to pyruvate by pyruvate kinase (PK). Oxaloacetate is reduced to malate by malate dehydrogenase (MDH). Fumarate hydratase (FH), alternatively termed fumarase, dehydrates malate to fumarate which is then converted by fumarate reductase (FR) to succinate. Finally, succinate is converted to propionate by as yet unknown enzymes. The conversion of fumarate to succinate depends on an electron chain (indicated by e⁻ and a wavy arrow) which might be involved in metronidazole reduction (Mtz to Mtz⁻). Importantly, also cytochrome ubiquinol oxidase depends on this electron chain. Pyruvate is converted to acetyl-CoA by pyruvate:ferredoxin oxidoreductase (PFOR) and/or pyruvate formate-lyase (PFL). Ultimately, acetyl-CoA is converted to acetate by phosphotransacetylase (PTA) and acetate kinase (AK). Alternatively, pyruvate can be reduced to lactate by lactate dehydrogenase (LDH). Lactate, however, is not a major end product under normal conditions. In the middle panel (ii) the altered metabolism of 638R^R is shown. Due to the lack of iron and/or heme, PFOR and FR are not functional. Further, PFL is strongly downregulated. Consequently, NAD+ must be recycled through the reduction of pyruvate and oxaloacetate, rendering lactate and malate the major metabolic end products. Pathways for the reduction of metronidazole are shut-down. The loss of cytochrome ubiquinol oxidase activity is compensated by upregulation of FprA. The presumptive metabolism of 638R nimA is shown in the right panel (iii). The formation of acetate is up due to increased activity of PFOR (indicated by enlarged letters). This is might be necessitated by a hypothetical interference of NimA (orange) with the electron chain which involved in the formation of succinate and could also lead to less metronidazole being reduced (inhibited pathway shown in gray). Importantly, this would also lead to a decreased activity of cytochrome ubiquinol oxidase, necessitating a compensatory upregulation of FprA. Finally, in order to ensure efficient regeneration of NAD+, more lactate could be formed in order to compensate for the decreased formation of succinate. Also malate might accumulate when formation of succinate is down.