Gene-Level Analysis

Introduction

Transcriptomes of FA1090, FA1090cpxA::kan, FA1090cpxR::erm mutant strains of Neisseria gonorrhoeae.

Dataset Description

RNA of Neisseria gonorrhoeae wildtype, cpxA, and cpxR mutants were collected at late log phase of growth, in quadruplicate (12 libraries in total).

Publication

Gangaiah D, Raterman EL, Wu H, Fortney KR, Gao H, Liu Y, Jerse AE, Spinola SM. Both MisR (CpxR) and MisS (CpxA) Are Required for Neisseria gonorrhoeae Infection in a Murine Model of Lower Genital Tract Infection. Infect Immun. 2017 Aug 18;85(9):e00307-17. doi: 10.1128/IAI.00307-17. PMID: 28652307; PMCID: PMC5563589.

 Abstract

During infection, Neisseria gonorrhoeae senses and responds to stress; such responses may be modulated by MisRS (NGO0177 and NGO0176), a two-component system that is a homolog of CpxRA. In Escherichia coli, CpxRA senses and responds to envelop stress; CpxA is a sensor kinase/phosphatase for CpxR, a response regulator. When a cpxA mutant is grown in medium containing glucose, CpxR is phosphorylated by acetyl phosphate but cannot be dephosphorylated, resulting in constitutive activation. Kandler and coworkers (J. L. Kandler, C. L. Holley, J. L. Reimche, V. Dhulipala, J. T. Balthazar, A. Muszyński, R. W. Carlson, and W. M. Shafer, Antimicrob Agents Chemother 60:4690–4700, 2016, https://doi.org/10.1128/AAC.00823-16 ) showed that MisR (CpxR) is required for the maintenance of membrane integrity and resistance to antimicrobial peptides, suggesting a role in gonococcal survival in vivo. Here, we evaluated the contributions of MisR and MisS (CpxA) to gonococcal infection in a murine model of cervicovaginal colonization and identified MisR-regulated genes using RNA sequencing (RNA-Seq). The deletion of misR or misS severely reduced the capacity of N. gonorrhoeae to colonize mice or maintain infection over a 7-day period and reduced microbial fitness after exposure to heat shock. Compared to the wild type (WT), the inactivation of misR identified 157 differentially regulated genes, most of which encoded putative envelope proteins. The inactivation of misS identified 17 differentially regulated genes compared to the WT and 139 differentially regulated genes compared to the misR mutant, 111 of which overlapped those differentially expressed in the comparison of the WT versus the misR mutant. These data indicate that an intact MisRS system is required for gonococcal infection of mice. Provided the MisR is constitutively phosphorylated in the misS mutant, the data suggest that controlled but not constitutive activation is required for gonococcal infection in mice.

Original Data

Bioinformatic Analysis

1- RNA-Seq Alignment

Application

RNA-Seq Alignments, BWA (Transcriptomics).

Input

Parameters

  • Input Reads: [Single End] SRR3666079-SRR3666090.fastq.gz files (12)

  • Upstream Files Pattern: _1

  • Downstream Files Pattern: _2

  • Genome Sequences: Neisseria_gonorrhoeae_fa_1090.ASM684v1.dna.toplevel.fa

  • Minimum Seed Length: 19

  • Band Width: 100

  • Z-dropoff: 100

  • Trigger Re-seeding: 1.5

  • Seed Occurrence: 20

  • Skip Seeds: 500

  • Drop Chains: 0.5

  • Discard Chains: 0

  • Mate Rescue Rounds: 50

  • Skip Mate Rescue: false

  • Skip Pairing: false

  • Matching Score: 1

  • Mismatch Penalty: 4

  • Gap Open Penalty (DEL): 6

  • Gap Open Penalty (INS): 6

  • Gap Extension Penalty (DEL): 1

  • Gap Extension Penalty (INS): 1

  • 5'-end Clipping Penalty: 5

  • 3'-end Clipping Penalty: 5

  • Unpaired Read Penalty: 17

  • Minimum Score: 30

  • Split Alignments as Primary: false

  • MapQ of Supp. Alignments: false

  • Output All Alignments: false

  • Soft Clipping for Supp.: false

  • Shorter Split Hits as Secondary: false

  • Sort BAM File By: Coordinates

Execution Time

10-15 minutes.

Output

2- Expression Quantification

Application

Create Count Table, Gene-level Quantification (Transcriptomics).

Input

Parameters

  • Input BAMs: SRR3666079-SRR3666090 BAM files

  • Input GFF/GTF: /00_reference/Neisseria_gonorrhoeae_fa_1090.ASM684v1.47.gtf

  • Quantification Level: gene

  • Name/Group By: gene_id:

  • Strand Specificity: Non Strand Specific

  • Overlap Mode: Union

  • Minimum Mapping Quality: 10

Execution Time

5-10 minutes.

Output:

3- Differential Expression Analysis

Application

Pairwise Differential Expression Analysis (Transcriptomics).

Input

Parameters

  • CPM Filter: 0.0

  • Samples reaching CPM Filter: 1

  • Normalization Method: TMM (Trimmed mean of M values)

  • Experimental Design File: experimental_design.txt

  • Design Type: Simple Design

  • Primary Experimental Factor: Strain

  • Primary Contrast Condition: FA1090cpxR

  • Primary Reference Condition: FA1090

  • Select a Statistical Test: Exact Test

  • Robust: true

Execution Time

5-10 minutes.

Output

4- Enrichment (GSEA)

Application

Gene Set Enrichment Analysis (Functional Analysis).

Input

Parameters

  • Reference Annotation: /00_reference/neisseria_gonorrhoeae_functions.box

  • FDR Filter for Ranked List: 1.0

  • Number of Permutations: 1000

  • Enrichment Statistic: Classic

  • Detailed Results for All GOs: false

  • Number of Detailed Results: 50

  • GO Categories: biological_process,molecular_function,cellular_component

  • Gene Sets Max Size: 500

  • Gene Sets Min Size: 15

  • Filter Mode: FDR

  • Filter Value: 0.25

Execution Time

1-2 minutes.

Output

5- Enrichment (Fisher’s Exact Test)

Application

Fisher’s Exact Test (Functional Analysis).

Input

Parameters

  • Reference Annotation: neisseria_gonorrhoeae_functions.annot

  • Test-Set Genes: Up-regulated genes

  • Do Not Filter: false

  • Filter Value: 0.05

  • Two Tailed: false

  • Remove Double IDs: false

  • Filter Mode: FDR

  • Annotations: GO IDs

  • GO Categories: biological_process,molecular_function,cellular_component

Execution Time

1-2 minutes.

Output:

Workflow

Gene-level analysis workflow.

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