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

Mariko Naito,Mikio Shoji,Janina P Lewis,Qin Gui,B Ross Belvin

doi:10.3390/microorganisms9030551

Mariko Naito, Mikio Shoji + Show 3 more

Open Access

https://doi.org/10.3390/microorganisms9030551

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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.

Highlights

The gene is 924 bp and codes for a 308-aa protein. This protein is predicted to play a role in the adaptation of P. intermedia to environmental stress as it is annotated as a peroxide activated regulator
Oxidative stress is the major variable in the oral cavity where the bacterium has to adapt to varying levels of atmospheric oxygen as well as to the oxidative stress released by the host immune defense mechanisms
P. intermedia isogenic mutantresistance placement of the the ErmF, providing through allelic replacement the OxyR-coding sequences for that reduced for macrolide–lincosamide in anaerobicof bacteria

Summary

Introduction

Prevotella intermedia is a highly abundant bacterium in the oral cavity. As shown in our recent publication, the genus Prevotella was one of the most abundant bacteria in biofilm samples collected from the subgingival pockets of patients with periodontal disease [1], as well as being prevalent in the salivary microbiome [2]. It is a black pigmented, anaerobic, Gram-negative bacterium, associated with periodontal disease, a destructive disease of the supporting structures of the teeth [3].

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