Human Serum-Specific Activation of Alternative Sigma Factors, the Stress Responders in Aggregatibacter actinomycetemcomitans

Gaoyan Tang-Siegel,Roger Bumgarner,Casey Chen,Teresa Ruiz,Weerayuth Kittichotirat,Weizhen Chen,Joyoti Basu

doi:10.1371/journal.pone.0160018

Abstract

Aggregatibacter actinomycetemcomitans, a known pathogen causing periodontal disease and infective endocarditis, is a survivor in the periodontal pocket and blood stream; both environments contain serum as a nutrient source. To screen for unknown virulence factors associated with this microorganism, A. actinomycetemcomitans was grown in serum-based media to simulate its in vivo environment. Different strains of A. actinomycetemcomitans showed distinct growth phenotypes only in the presence of human serum, and they were grouped into high- and low-responder groups. High-responders comprised mainly serotype c strains, and showed an unusual growth phenomenon, featuring a second, rapid increase in turbidity after 9-h incubation that reached a final optical density 2- to 7-fold higher than low-responders. Upon further investigation, the second increase in turbidity was not caused by cell multiplication, but by cell death. Whole transcriptomic analysis via RNA-seq identified 35 genes that were up-regulated by human serum, but not horse serum, in high-responders but not in low-responders, including prominently an alternative sigma factor rpoE (σE). A lacZ reporter construct driven by the 132-bp rpoE promoter sequence of A. actinomycetemcomitans responded dramatically to human serum within 90 min of incubation only when the construct was carried by a high responder strain. The rpoE promoter is 100% identical among high- and low-responder strains. Proteomic investigation showed potential interactions between human serum protein, e.g. apolipoprotein A1 (ApoA1) and A. actinomycetemcomitans. The data clearly indicated a different activation process for rpoE in high- versus low-responder strains. This differential human serum-specific activation of rpoE, a putative extra-cytoplasmic stress responder and global regulator, suggests distinct in vivo adaptations among different strains of A. actinomycetemcomitans.

Highlights

The gram-negative, non-motile, capnophilic bacterium Aggregatibacter actinomycetemcomitans is a recognized oropharyngeal colonizer, found in the oral cavity of 20% of the population [1]
Distinct growth phenotypes of A. actinomycetemcomitans were only identified in the presence of human serum
The high-responder strains showed a growth phenomenon, characterized by an initial logarithmic phase starting at 3–4 h, followed by a second rapid increase of turbidity 9 h after exposure to human serum, which reached a final optical density (OD) 2- to 7-fold higher than the low-responder strains grown under the same conditions (Fig 1A)

Summary

Introduction

The gram-negative, non-motile, capnophilic bacterium Aggregatibacter actinomycetemcomitans is a recognized oropharyngeal colonizer, found in the oral cavity of 20% of the population [1]. Our earlier work though comparative genomic analysis identified 3,301 genes in the pangenome of A. actinomycetemcomitans, including 2034 “core genes” that are present in all strains and 1,267 “accessory genes” that are not present in all strains of this species [11]. [11]In addition, certain strain of A. actinomycetemcomitans, e.g. D11S-1, carries large plasmids and is infected by bacteriophages [14]. Such extra genetic information carried by plasmids and bacteriophages may change bacterial growth fitness and virulence [15, 16]. We hypothesize that accessory genes and mobile genetic elements including plasmids and phages modulate the fitness and virulence of A. actinomycetemcomitans in vivo, eventually leading to disparate consequences of infection by this organism in human hosts

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