uses a single homodimeric disulfide bond (DSB) oxidoreductase DsbA2 to catalyze extracytoplasmic protein folding and to correct DSB errors through protein-disulfide isomerase (PDI) activity. cycle between an intracellular vegetative replicating form and an extracellular metabolically dormant cyst-like form or MIF for mature intracellular form (Garduno et al. 2002 Cyst germination appears to be restricted to intracellular environments by an obligate requirement for the amino acid cysteine believed to be scarce in natural environments (Tison et al. 1980 Ewann et al. 2006 Restricted germination may also be important in the transmission of disease to humans as Legionnaires’ disease is more prevalent when environmental conditions are permissive to growth of natural amoebic hosts that amplify and concentrate cysts. Proteomic profiling identified proteins enriched for in cysts one of which is a novel homodimeric disulfide bond (Dsb) oxidoreductase (DsbA2) that is phylogenetically distinct from the DsbA oxidases and DsbC protein disulfide isomerase (PDI)/reductases so well studied in (Jameson-Lee et al. 2011 A 6-fold increase in ASP9521 transcript levels in cysts when compared to levels from exponentially growing bacteria suggested that DsbA2 may be developmentally regulated and a participant in the extensive remodeling of the cell envelope during differentiation (Kpadeh et al 2013 Rabbit polyclonal to ACSS2. While DsbA function in other bacteria is required for motility and virulence (Heras et al. 2009 we showed that mutants of were indistinguishable from wild-type parental strains for infectivity (amoeba and HeLa cell models) and motility; and were essentially without correlating phenotypes suggesting that DsbA2 likely plays a prominent role in managing disulfide bonding and protein folding (Jameson-Lee et al. 2011 These studies also showed that expression of a mutant DsbA2(P198T) protein in produced a dominant negative effect on DsbA2 ASP9521 function resulting in loss of motility and infectivity both of which ASP9521 are important for pathogenesis (Berger and Isberg 1993 Brand et al. 1994 Molofsky et al. 2005 We traced the loss of infectivity to the Dot/Icm Type IVb secretion system (T4SS) by showing that DsbA2(P198T) captured structural proteins (DotG DotC DotK and IcmX) of the core complex (Jameson-Lee et al. 2011 Kubori et al. 2014 The Dot/Icm T4SS is required for virulence and delivery of nearly 300 cytoplasmic effector proteins into host cells (Chen et al. 2004 Luo et al. 2004 Huang et al. 2011 Zhu et al. 2011 Lifshitz et al. 2013 DsbA2 is not unique to the legionellae but is widely distributed among Gram negative bacteria that lack orthologues of DsbC and DsbG and is highly conserved among ASP9521 other intracellular human animal and plant pathogens that employ T4SSs for virulence (Coxiella Anaplasma Rickettesiae Brucella Agrobacterium Bartonella and (Bardwell et al. 1991 DsbA catalyzes consecutive disulfide bond formation and the disulfide is regenerated through oxidation by cytoplasmic membrane protein DsbB that delivers obtained reducing equivalents to a quinone cofactor associated with electron transport and respiration (Bardwell et al. 1991 Zapun et al. 1993 Wunderlich et al. 1993 Collet and Bardwell 2002 For proteins requiring nonconsecutive disulfide bonding or those containing inappropriate disulfides proper folding or repair requires both reduction and isomerization of disulfides that is catalyzed by homodimeric DsbC (Collet et al. 2002 Berkman et al. 2005 Ren & Bardwell 2011 DsbC is maintained in the free thiol form by DsbD a transmembrane protein that shuttles reducing equivalents from the cytoplasmic NADPH ASP9521 thioredoxin-thioredoxin reductase system through an intermolecular thiol exchange process within DsbD (Zapun et al. 1995 Depuydt et al. 2009 Cho and Beckwith 2009 Depuydt et al. 2011 Extensive studies of the DsbA and DsbC systems in show that these systems are mutually exclusive and do not interact (Bader et al. 2001 Segatori et al. 2004 Kadokura et al. 2004 Kadokura and Beckwith 2010 It seems ASP9521 obvious that if DsbC were to be oxidized by DsbA or by DsbB that the reducing equivalents obtained from cytoplasmic pools of NADPH would be frittered away via respiration creating a futile cycle. We refer to the DSB system as a two-player system where DsbA and DsbC systems do not interact. In contrast the DsbA2 system of appears to be a single player system in which homodimeric DsbA2 provides both DsbA oxidase and DsbC protein disulfide isomerase (PDI) functions (Jameson-Lee et al. 2011 and Kpadeh et al. 2013 An important clue as to how a single player system might.