Comparison of inherently essential genes of Porphyromonas gingivalis identified in two transposon‐sequencing libraries

Abstract

Summary Porphyromonas gingivalis is a Gram-negative anaerobe and keystone periodontal pathogen. A mariner transposon insertion mutant library has recently been used to define 463 genes as putatively essential for the in vitro growth of P. gingivalis ATCC 33277 in planktonic culture [Library 1, (Klein et al 2012)]. We have independently generated a transposon insertion mutant library [Library 2] for the same P. gingivalis strain and herein compare genes that are putatively essential for in vitro growth in complex media, as defined by both libraries. 281 genes (61%) identified by Library 1 were common to Library 2. Many of these common genes are involved in fundamentally important metabolic pathways, notably pyrimidine cycling as well as lipopolysaccharide, peptidoglycan, pantothenate and coenzyme A biosynthesis and nicotinate and nicotinamide metabolism. Also in common are genes encoding heat shock protein homologues, sigma factors, enzymes with proteolytic activity and the majority of sec-related protein export genes. In addition to facilitating a better understanding of critical physiological processes, transposon sequencing technology has the potential to identify novel strategies for the control of P. gingivalis infections. Those genes defined as essential by two independently generated TnSeq mutant libraries are likely to represent particularly attractive therapeutic targets.

Genes Contributing to Porphyromonas gingivalis Fitness in Abscess and Epithelial Cell Colonization Environments

“…The abscess model will identify mutants with a deficiency in survival in a more complex environment which contains host immune factors. Core essential genes required for in vitro growth of P. gingivalis have been identified in our previous study (Hutcherson et al, 2016 ), and these were not considered in the data analysis. In the epithelial cell colonization model, 498 genes were determined conditionally essential, whereas 545 genes were determined essential in the abscess group, using a stringent log2 cutoff of >3.3 which represents a 10-fold difference.…”

“…The construction of the transposon library was previously described by Hutcherson et al ( 2016 ). Briefly, a saturated transposon library was generated using a mariner transposon system (Goodman et al, 2009 ) in P. gingivalis 33277.…”

“…Libraries for sequencing were constructed as described previously (Hutcherson et al, 2016 ). Double-stranded, barcoded, DNA adapters were created using the LIB_Adapt primers (_control, _TIGK or _abscess) (Supplementary Table 1 ) to differentiate sequencing groups in the same flow lane.…”

“…Characteristics of the library are provided in Supplementary Table 2 . Reads were counted and normalized to reads per kilobase of transcript per million reads mapped (RPKM), and genes with a RPKM <5 were considered inherently essential (Klein et al, 2012 ; Hutcherson et al, 2016 ) and not considered further. The number of reads for each gene in the input pool (i.e., the library of P. gingivalis transposon mutants used to inoculate TIGKs or mice) was compared to the number of reads in the equivalent gene in the two output pools (i.e., the library of transposon mutants recovered after selection in mice or in TIGKs) to calculate fold change.…”

“…Transposon mutagenesis can create a large pool of highly-saturated mutant libraries, and the comparative contributions of bacterial genes can be assessed following selection for fitness in different environments. In this study, we utilized a P. gingivalis Mariner based Tn library (Hutcherson et al, 2016 ) to perform an unbiased search for genes involved in epithelial cell interactions and in vivo survival in a murine abscess model. Genes encoding previously unrecognized properties that make measurable contributions to survival in these contexts were selected for further analysis by targeted gene disruption.…”