Sec-mediated secretion by Coxiella burnetii

Abstract

BackgroundCoxiella burnetii is a Gram-negative intracellular bacterial pathogen that replicates within a phagolysosome-like parasitophorous vacuole (PV) of macrophages. PV formation requires delivery of effector proteins directly into the host cell cytoplasm by a type IVB secretion system. However, additional secretion systems are likely responsible for modification of the PV lumen microenvironment that promote pathogen replication.ResultsTo assess the potential of C. burnetii to secrete proteins into the PV, we analyzed the protein content of modified acidified citrate cysteine medium for the presence of C. burnetii proteins following axenic (host cell-free) growth. Mass spectrometry generated a list of 105 C. burnetii proteins that could be secreted. Based on bioinformatic analysis, 55 proteins were selected for further study by expressing them in C. burnetii with a C-terminal 3xFLAG-tag. Secretion of 27 proteins by C. burnetii transformants was confirmed by immunoblotting culture supernatants. Tagged proteins expressed by C. burnetii transformants were also found in the soluble fraction of infected Vero cells, indicating secretion occurs ex vivo. All secreted proteins contained a signal sequence, and deletion of this sequence from selected proteins abolished secretion. These data indicate protein secretion initially requires translocation across the inner-membrane into the periplasm via the activity of the Sec translocase.ConclusionsC. burnetii secretes multiple proteins, in vitro and ex vivo, in a Sec-dependent manner. Possible roles for secreted proteins and secretion mechanisms are discussed.

Distinctive Genome Reduction Rates Revealed by Genomic Analyses of TwoCoxiella-Like Endosymbionts in Ticks

“…Additionally, all genes associated with the type IV pili are pseudogenes in CRt and absent in CLEAA. Although a pilus has not been observed in C. burnetii , this system has been speculated to participate in secretion of proteins translocated to the periplasmic space through the Sec system ( Stead et al 2013 ) as seen in Francisella novocida ( Hager et al 2006 ). Together, these losses suggest that neither CRt nor CLEAA are residing in phagosome-derived vacuoles like C. burnetii but instead are enclosed in another perhaps less hostile host-derived membrane compartment ( Lalzar et al 2014 ), as seen in other obligate mutualistic bacteria ( Moran and Bennett 2014 ).…”

Right on Q: Genetics begin to Unravel Coxiella Burnetii host cell Interactions

“…Evidence for host membrane contact is based on the observation that verified effector proteins are not detected by mass spectrometry in ACCM-2 following Coxiella culture [136] and electron micrographs showing protrusions emanating from the Coxiella surface that appear to penetrate the CCV lipid bilayer (Figure 3). The presence of Dot/Icm proteins with Walker box ATPase motifs, such as DotB, suggests substrate translocation is an energy-requiring process that requires active pathogen metabolism [84].…”

Cloning, expression, and characterization of a Coxiella burnetii Cu/Zn Superoxide dismutase

“…The C. burnetii genomic database from TIGR predicted a putative Cu/Zn SOD with a signal sequence (CBU 1822). Recently, Stead et al reported the presence of a putative SodC protein in supernatants from C. burnetii cultured in acidified citrate cysteine media using a FLAG-tag assay, which indicates the secreted nature of the SOD [ 5 ]. Periplasmically localized Cu/Zn SOD enzymes have been identified in a wide range of Gram-negative bacteria and are proposed to protect bacteria from exogenously produced toxic oxygen radicals, which indicate the potential significance of a C. burnetii Cu/Zn SOD.…”