Francisella tularensis RipA Protein Topology and Identification of Functional Domains

Abstract

Francisella tularensis is a Gram-negative coccobacillus and is the etiological agent of the disease tularemia. Expression of the cytoplasmic membrane protein RipA is required for Francisella replication within macrophages and other cell types; however, the function of this protein remains unknown. RipA is conserved among all sequenced Francisella species, and RipA-like proteins are present in a number of individual strains of a wide variety of species scattered throughout the prokaryotic kingdom. Crosslinking studies revealed that RipA forms homoligomers. Using a panel of RipA-green fluorescent protein and RipA-PhoA fusion constructs, we determined that RipA has a unique topology within the cytoplasmic membrane, with the N and C termini in the cytoplasm and periplasm, respectively. RipA has two significant cytoplasmic domains, one composed roughly of amino acids 1 to 50 and the second flanked by the second and third transmembrane domains and comprising amino acids 104 to 152. RipA functional domains were identified by measuring the effects of deletion mutations, amino acid substitution mutations, and spontaneously arising intragenic suppressor mutations on intracellular replication, induction of interleukin-1␤ (IL-1␤) secretion by infected macrophages, and oligomer formation. Results from these experiments demonstrated that each of the cytoplasmic domains and specific amino acids within these domains are required for RipA function.

Extragenic suppressor mutations in ΔripA disrupt stability and function of LpxA

“…RipA is a cytoplasmic membrane protein containing two domains that are essential for function and are accessible to the cytoplasm [ 2 ]. LpxA is a soluble protein that interacts with acyl-ACP on the cytoplasmic membrane.…”

“…RipA is dispensable for growth in minimal media, but is required for F. tularensis virulence in mouse models of infection [ 1 ]. RipA is a homooligomeric cytoplasmic membrane protein that contains two cytoplasmic domains that are essential for RipA function [ 2 ]. The ∆ ripA strain infects host cells and escapes the phagosome entering the cytoplasm similar to wild type F. tularensis , but fails to replicate within the cytosol [ 1 ].…”

“…Determining RipA function would provide an important insight into the specific mechanisms used by F. tularensis to grow within eukaryotic cells. Given the scant information obtained from bioinformatics analysis of RipA, and the eclectic group of organisms that express RipA-like proteins [ 2 ], we took genetic and biochemical approaches to determine RipA function. We isolated independent extragenic suppressor mutations of ∆ ripA that restored intracellular growth, and all suppressor mutations mapped to either lpxA or glmU .…”

Identifying Francisella tularensis Genes Required for Growth in Host Cells

“…Since there is only one predicted transmembrane domain (TMHMM) at the extreme N terminus, the majority of the protein must be located within the cytoplasm or periplasm. To discern between these two possibilities, we adapted a GFP/PhoA fusion system to function in Francisella species (27). Constructs expressing translational fusions of GFP or PhoA to the C terminus of FTT_0924 were constructed and expressed in F. tularensis Schu S4.…”

“…All fractions were standardized to equal protein by using a bicinchoninic acid (BCA) assay (Pierce), run on a 4 to 20% polyacrylamide gradient gel (Bio-Rad), and transferred to a nitrocellulose membrane. The membranes were probed with antibodies recognizing hemagglutinin (HA) peptide (Sigma), RipA (27), IglC (BEI Resources), and Tul4 (BEI Resources) for primary probes and antibodies conjugated to fluorophores as secondary probes (LI-COR Biosciences). Blots were visualized with an Odyssey infrared imaging system (LI-COR Biosciences).…”