Insertional Inactivation of Prevotella intermedia OxyR Results in Reduced Survival with Oxidative Stress and in the Presence of Host Cells

Abstract

One of the most abundant bacteria in the subgingival pockets of patients with bleeding following mechanical periodontal therapy is Prevotella intermedia. However, despite its abundance, the molecular mechanisms of its contribution to periodontal disease are not well known. This is mainly due to the lack of genetic tools that would allow examination of the role of predicted virulence factors in the pathogenesis of this bacterium. Here, we report on the first mutant in the P. intermedia OMA14 strain. The mutation is an allelic exchange replacement of the sequences coding for a putative OxyR regulator with ermF sequences coding for the macrolide–lincosamide resistance in anaerobic bacteria. The mutant is severely impaired in its ability to grow with eukaryotic cells, indicating that it is an important target for interventional strategies. Further analyses reveal that its ability to grow with oxidative stress species, in the form of hydrogen peroxide and oxygen, is severely affected. Transcriptome analysis reveals that the major deregulated genes code for the alkylhydroperoxide reductase system, AhpCF, mediating protection from peroxide stress. Moreover, genes coding for Dps, CydA and Ftn are downregulated in the mutant strain, as further verified using qRT-PCR analysis. In conclusion, we succeeded in generating the first P. intermedia mutant and show that the OxyR-deficient strain is unable to survive with a variety of host cells as well as with oxidative stress.

The oxidoreductase activity of Rnf balances redox cofactors during fermentation of glucose to propionate in Prevotella

“…Genetic manipulation systems for Prevotella species in this study have not been built due to limited genetic tools available for this group 41 . Recently, great progress has been made in Prevotella intermedia 42 , P. copri 43 , and other Prevotella 44 , which can promote development of new genetic tools for P. brevis and P. ruminicola . Despite the lack of genetic analysis of Rnf in Prevotella species, similar genetic evidence demonstrating that Rnf has ferredoxin:NAD + oxidoreductase activity have been shown in several prokaryotes, such as Methanosarcina acetivorans 45 , B. fragilis 28 , and Clostridium thermocellum 46 .…”

Iron Deficiency Modulates Metabolic Landscape of Bacteroidetes Promoting Its Resilience during Inflammation

“…Raw counts, which in turn were normalized and transformed, were used in the analysis. Two-group unpaired comparisons were carried out using reads per kilobase per million (RPKM) (RPKM is defined as the number of reads that are assigned to the transcript and divided by the transcript length and normalized by mapped reads) values ( 64 ). The t test was used to carry out the statistical analysis for the RNAseq data.…”

Iron deficiency modulates metabolic landscape of Bacteroidetes promoting its resilience during inflammation

“…The RNA isolation and library preparation were done as described previously 19, 20 . Briefly, the RNeasy mini-kit (Qiagen) was used to isolate RNA from bacterial pellets following manufacturer’s protocol.…”

“…Thus, resulting transcriptome-derived sequences were aligned to the reference genomes of: P. gingivalis W83 (for P. gingivalis W83 transcriptome), P. gingivalis ATCC33277 (for P. gingivalis ATCC33277 transcriptome), P. intermedia OMA14 (for the P. intermedia OMA14 transcriptome) and to B. thetaiotaomicron VPI BT5482 (for the B. thetaiotaomicron VPI BT5482 Δ tdk transcriptome). The enrichment for the iron-dependent regulation was determined by dividing the number of reads derived from the bacteria grown in iron-deplete conditions to that derived from the bacteria grown under iron replete conditions for each position of the genome following normalization by aligned fragment per kilobase of transcript per million (FPKM) 21, 20, 19 .…”