Increased leukotoxin production: Characterization of 100 base pairs within the 530 base pair leukotoxin promoter region of Aggregatibacter actinomycetemcomitans

Vandana Sampathkumar,Daniel H Fine,Dipti Godboley,Senthil Kumar Velusamy

doi:10.1038/s41598-017-01692-6

Vandana Sampathkumar, Daniel H Fine + Show 2 more

Open Access

https://doi.org/10.1038/s41598-017-01692-6

Copy DOI

Journal: Scientific Reports	Publication Date: May 15, 2017
Citations: 17	License type: open-access

Affiliation: Rutgers, The State University of New Jersey

Abstract

Aggregatibacter actinomycetemcomitans leukotoxin (LtxA) is a major virulence factor that kills leukocytes permitting it’s escape from host immune surveillance. A. actinomycetemcomitans strains can produce high or low levels of toxin. Genetic differences reside in the “so called JP2” ltxA promoter region. These hyper-leukotoxin producing strains with the 530 bp deletion have been studied in detail. However, regions contained within the 530 bp deletion that could be responsible for modulation of leukotoxin production have not been defined. Here, we report, for the first time, on regions within the 530 bp that are responsible for high-levels of ltxA expression. We constructed a deletion of 530 bps in a primate isolate of A. actinomycetemcomitans, which produced leukotoxin equivalent to the JP2 strain. We then constructed sequential deletions in regions that span the 530 bps. Results indicated that expression of the ltxA transcript was reduced by a potential transcriptional terminator in promoter region 298 to 397 with a ΔG = −7.9 kcal/mol. We also confirmed previous findings that transcriptional fusion between the orfX region and ltxC increased ltxA expression. In conclusion, we constructed a hyper-leukotoxin producing A. actinomycetemcomitans strain and identified a terminator located in the promoter region extending from 298–397 that alters ltxA expression.

Highlights

Aggregatibacter actinomycetemcomitans leukotoxin (LtxA) is a major virulence factor that kills leukocytes permitting it’s escape from host immune surveillance
Since these studies were conducted in the JP2-like strain they focused on the role of regulatory proteins that might bind to DNA upstream from the missing promoter region[5, 6, 10]
Our principal goal is to study the role of different virulence factors of A. actinomycetemcomitans in vivo in a real world Rh monkey model

Summary

Introduction

Aggregatibacter actinomycetemcomitans leukotoxin (LtxA) is a major virulence factor that kills leukocytes permitting it’s escape from host immune surveillance. At the genetic level the hyper-producing strain shows a deletion of 530 bp in the promoter region that appears to be responsible for increased expression of downstream ltx genes[4]. Studies have shown that this deletion appears to, 1) upregulate ltxA expression, and 2) result in differential transcription that can influence ltxA expression These studies have been performed, for the most part, in JP2-like strains that; 1) lack many characteristics seen in recently isolated clinical isolates, and 2) are devoid of the 530 bp promoter region thought to be critical for elevated leukotoxin production[8]. Most comparisons are based on speculation and abductive inference[15] Since these studies were conducted in the JP2-like strain they focused on the role of regulatory proteins that might bind to DNA upstream from the missing promoter region[5, 6, 10]. We found that a key determinant for expression of leukotoxin is found in a 100 bp sequence in the promoter region that contains a terminator, which when deleted permits high levels of production

Objectives

Methods

Results

Conclusion