Large-Scale Analysis of the Mycoplasma bovis Genome Identified Non-essential, Adhesion- and Virulence-Related Genes

Abstract

Mycoplasma bovis is an important pathogen of cattle causing bovine mycoplasmosis. Clinical manifestations are numerous, but pneumonia, mastitis, and arthritis cases are mainly reported. Currently, no efficient vaccine is available and antibiotic treatments are not always satisfactory. The design of new, efficient prophylactic and therapeutic approaches requires a better understanding of the molecular mechanisms responsible for M. bovis pathogenicity. Random transposon mutagenesis has been widely used in Mycoplasma species to identify potential gene functions. Such an approach can also be used to screen genomes and search for essential and non-essential genes for growth. Here, we generated a random transposon mutant library of M. bovis strain JF4278 containing approximately 4000 independent insertion sites. We then coupled high-throughput screening of this mutant library to transposon sequencing and bioinformatic analysis to identify M. bovis non-essential, adhesion- and virulence-related genes. Three hundred and fifty-two genes of M. bovis were assigned as essential for growth in rich medium. Among the remaining non-essential genes, putative virulence-related factors were subsequently identified. The complete mutant library was screened for adhesion using primary bovine mammary gland epithelial cells. Data from this assay resulted in a list of conditional-essential genes with putative adhesion-related functions by identifying non-essential genes for growth that are essential for host cell-adhesion. By individually assessing the adhesion capacity of six selected mutants, two previously unknown factors and the adhesin TrmFO were associated with a reduced adhesion phenotype. Overall, our study (i) uncovers new, putative virulence-related genes; (ii) offers a list of putative adhesion-related factors; and (iii) provides valuable information for vaccine design and for exploring M. bovis biology, pathogenesis, and host-interaction.

Keywords: Mycoplasma bovis; adhesion; non-essential genes; random transposon mutagenesis; virulence.

FIGURE 1

Schematic representation of the adhesion screening and bioinformatics analyses. The adhesion screening was performed with the full mutant library containing approximately 4000 mutants (input sample). After adhesion, adherent mutants (output sample) were recovered. The screening of the same full random transposon mutant library was performed in three replicates, leading to the collection of three input samples and three output samples. Transposon sequencing was carried out by parallel sequencing of the regions flanking each transposon to identify disrupted genes. Transposons sequencing read data from input samples from triplicates were used to categorize genes as essential and non-essential for growth on rich medium using the ESSENTIALS software (Zomer et al., 2012). Transposons sequencing read data of input and output samples from triplicates were then compared to identify conditional-essential genes (essential for adhesion) using the ESSENTIALS software (Zomer et al., 2012).

FIGURE 2

Location of transposon insertion sites in the genome of M. bovis strain JF4278. Genes and their orientation are indicated by arrows. Genes found essential with ESSENTIALS are filled in red, while non-essential genes are filled in yellow (Zomer et al., 2012). Mapping of the transposon integrations sites from the transposon sequencing of the full mutant library (input samples) is represented by black, green, and red bars (each corresponding to one replicate experiment). The height of the insertion bars represents the read count per insertion. Approximately 4000 independent transposon insertion sites were detected with an average sequencing depth (reads/transposon insertion) of 6800 per transposon-junction per replicate.

FIGURE 3

Density plot of essentiality values obtained with the software ESSENTIALS. Read counts per gene of the input samples were compared to the expected number of reads per gene calculated by ESSENTIALS (Zomer et al., 2012). M. bovis strain JF4278 genes are assigned a fold change (FC) value of read counts in log2 scale compared to the expected number of reads per gene. Log2(FC) values (essentiality values) for all genes are shown on the x-axis of the density plot. Essentiality values show a bimodal distribution. The log2(FC) value of non-essential JF4278 genes is around zero. Genes more likely to be essential have negative log2(FC) values. The cut-off value for essential genes suggested by the software ESSENTIALS for this data set is log2(FC) < –4.64.

FIGURE 4

KEGG pathway enrichment analysis of essential genes in strain JF4278. Different KEGG pathways from M. bovis, with the corresponding mbov ID number, are displayed. Significant (P-value ≤ 0.01) overrepresented KEGG pathways in the essential gene set compared to the complete gene set of strain JF4278 are shown. Gray columns correspond to the P-value for significance of enrichment in –log10 transformation. Spotted columns correspond to the fold enrichment of individual KEGG pathways between complete and essential gene set. Significance of enrichment was calculated with the Fisher’s exact test.

FIGURE 5

Adhesion assay with the full M. bovis strain JF4278 mutant library. M. bovis adhesion to bMec cells after 30 min. The y-axis represents the percentage of adherent M. bovis relative to the added M. bovis, calculated by using the number of CFUs counted from input samples and output samples. Adhesion assay with the full M. bovis mutant library was performed in three independent experiments indicated by black dots. Median values are indicated as horizontal lines.

FIGURE 6

Density plot of conditional-essentiality values obtained with the software ESSENTIALS. Read counts per gene of output samples were compared to data from input samples by ESSENTIALS (Zomer et al., 2012). M. bovis strain JF4278 genes from output samples are assigned a fold change (FC) value of read counts in log2 scale compared to the read counts of input samples. Log2(FC) values for all genes are shown on the x-axis of the density plot. Log2(FC) values show a bimodal distribution. The log2(FC) value of non-essential JF4278 genes is around zero. Genes more likely to be conditional-essential (essential for adhesion) have negative log2(FC) values. The cut-off value for conditional-essential genes suggested by the software ESSENTIALS for this data set is log2(FC) < –3.38.

FIGURE 7

Overview of non-essential, essential, and conditional-essential genes in the genome of M. bovis strain JF4278. Genes and their orientation are indicated by arrows. Genes found essential with ESSENTIALS are filled in red, while non-essential genes are filled in yellow (Zomer et al., 2012). Genes found to be conditional-essential (essential for adhesion) are filled in purple. Essentiality was assessed with transposon sequencing data of the full mutant library (input samples) with ESSENTIALS. Conditional-essentiality was assessed by comparing input samples to output samples with ESSENTIALS. Mapping of the transposon integrations sites from the transposon sequencing of the input samples (Inputs) and output samples (Outputs) are represented by black, green, and red bars (each corresponding to one replicate). The height of the insertion bars represents the read count per insertion.

FIGURE 8

Adhesion assay of individual candidate mutants of M. bovis strain JF4278. M. bovis adhesion to bMec cells after 30 min. The y-axis represents the percentage of adherent M. bovis relative to the added M. bovis. Adhesion with the wild-type strain or single mutants was performed in four independent experiments as indicated by black dots. Median values are indicated as horizontal lines in the box plot. Statistical comparison of each mutant to the wild-type strain was performed by the two-sample Wilcoxon test. ^∗P < 0.05.