Essential genes for Haemophilus parainfluenzae survival and biofilm growth

Abstract

Haemophilus parainfluenzae (Hp) is a Gram-negative, highly prevalent, and abundant commensal in the human oral cavity, and an infrequent extraoral opportunistic pathogen. Hp occupies multiple niches in the oral cavity, including the supragingival plaque biofilm. Little is known about how Hp interacts with its neighbors in healthy biofilms nor its mechanisms of pathogenesis as an opportunistic pathogen. To address this, we identified the essential genome and conditionally essential genes in in vitro biofilms aerobically and anaerobically. Using transposon insertion sequencing (TnSeq) with a highly saturated mariner transposon library in two strains, the ATCC33392 type-strain (Hp 392) and oral isolate EL1 (Hp EL1), we show that the essential genomes of Hp 392 and Hp EL1 are composed of 395 (20%) and 384 (19%) genes, respectively. The core essential genome, consisting of 341 (17%) essential genes conserved between both strains, was composed of genes associated with genetic information processing, carbohydrate, protein, and energy metabolism. We also identified conditionally essential genes for aerobic and anaerobic biofilm growth, which were associated with carbohydrate and energy metabolism in both strains. RNAseq analysis determined that most genes upregulated during anaerobic growth are not essential for Hp 392 anaerobic survival. The completion of this library and analysis under these conditions gives us a foundational insight into the basic biology of H. parainfluenzae in differing oxygen conditions, similar to its in vivo habitat. This library presents a valuable tool for investigation into conditionally essential genes for an organism that lives in close contact with many microbial species in the human oral habitat.IMPORTANCEHaemophilus parainfluenzae is a highly abundant human commensal microbe, present in most healthy individuals where it colonizes the mouth. H. parainfluenzae correlates with good oral health and may play a role in preservation of healthy host status. Also, H. parainfluenzae can cause opportunistic infections outside of the oral cavity. To date, little is known about how H. parainfluenzae colonizes the human host, despite being such a frequent and abundant part of our human microbiome. Here, we demonstrate the creation and use of a powerful tool, a TnSeq library, used to identify genes necessary for both the outright growth of this organism and also genes conditionally essential for growth in varying oxygen status which it can encounter in the human host. This tool and these data serve as a foundation for further study of this relatively unknown organism that may play a role in preserving human health.

Keywords: Haemophilus; TnSeq; biofilms; commensal; dental plaque; facultative anaerobes; oral microbiology.

Fig 1

The essential genome of Hp 392 (A) and Hp EL1 (B). The outer (purple) ring indicates transposon insertions, next inward (gray) represents open reading frames, and the inner ring (green) indicates essential genes. Figure (C) shows the log2 fold change (FC) in mutant abundance for Hp 392 (x-axis) versus EL1 (y-axis). Each point corresponds to an ortholog and genes <200 bp were excluded from the analysis.

Fig 2

KEGG group assignments for absolute essential genes in Hp. The essential genome of Hp 392 (A) and Hp EL1 (B) grouped into KEGG categories. Genes <200 bp were not included in the analysis. Most essential genes are involved in central cell functional processes including genetic information processing and common energy metabolism pathways.

Fig 3

The core essential genome of Hp. Comparison of 18 genomes of Hp and the core essential genome of Hp ATCC 392 and Hp EL1 (top two rows) with the full genomes and the 16 available Hp genomes. Purple indicates the core essential genome, that is, the TnSeq identified absolute essential genes that are conserved in all species.

Fig 4

Genes essential for aerobic and anaerobic growth in a colony biofilm model for Hp 392 and Hp EL1. Conditionally essential genes for aerobic and anaerobic growth in in vitro biofilms in Hp 392 (blue), and Hp EL1 (yellow) grouped into higher KEGG functions.

Fig 5

The majority of Hp 392 genes conditionally essential for anaerobic biofilm growth are not differentially regulated between aerobic and anaerobic conditions. Ninety-eight genes, indicated in blue, were significantly upregulated (P ≤ 0.05, fold change ≥2) in anaerobic compared to aerobic conditions, while 67 genes, indicated in red, were significantly downregulated (P ≤ 0.05, fold change ≤−2). Genes uniquely essential for colony growth under anaerobic conditions are indicated in black with only three total being significantly differentially expressed.