BB0324 and BB0028 are constituents of the Borrelia burgdorferi β-barrel assembly machine (BAM) complex

<p>Abstract</p> <p>Background</p> <p>Similar to Gram-negative bacteria, the outer membrane (OM) of the pathogenic spirochete, <it>Borrelia burgdorferi</it>, contains integral OM-spanning proteins (OMPs), as well as membrane-anchored lipoproteins. Although the mechanism of OMP biogenesis is still not well-understood, recent studies have indicated that a heterooligomeric OM protein complex, known as BAM (β-barrel assembly machine) is required for proper assembly of OMPs into the bacterial OM. We previously identified and characterized the essential β-barrel OMP component of this complex in <it>B. burgdorferi</it>, which we determined to be a functional BamA ortholog.</p> <p>Results</p> <p>In the current study, we report on the identification of two additional protein components of the <it>B. burgdorferi </it>BAM complex, which were identified as putative lipoproteins encoded by ORFs BB0324 and BB0028. Biochemical assays with a BamA-depleted <it>B. burgdorferi </it>strain indicate that BB0324 and BB0028 do not readily interact with the BAM complex without the presence of BamA, suggesting that the individual <it>B. burgdorferi </it>BAM components may associate only when forming a functional BAM complex. Cellular localization assays indicate that BB0324 and BB0028 are OM-associated subsurface lipoproteins, and <it>in silico </it>analyses indicate that BB0324 is a putative BamD ortholog.</p> <p>Conclusions</p> <p>The combined data suggest that the BAM complex of <it>B. burgdorferi </it>contains unique protein constituents which differ from those found in other proteobacterial BAM complexes. The novel findings now allow for the <it>B. burgdorferi </it>BAM complex to be further studied as a model system to better our understanding of spirochetal OM biogenesis in general.</p

Identification of a novel transport system in Borrelia burgdorferi that links the inner and outer membranes

Borrelia burgdorferi, the spirochete that causes Lyme disease, is a diderm organism that is similar to Gram-negative organisms in that it contains both an inner and outer membrane. Unlike typical Gram-negative organisms, however, B. burgdorferi lacks lipopolysaccharide (LPS). Using computational genome analyses and structural modeling, we identified a transport system containing six proteins in B. burgdorferi that are all orthologs to proteins found in the lipopolysaccharide transport (LPT) system that links the inner and outer membranes of Gram-negative organisms and is responsible for placing LPS on the surface of these organisms. While B. burgdorferi does not contain LPS, it does encode over 100 different surface-exposed lipoproteins and several major glycolipids, which like LPS are also highly amphiphilic molecules, though no system to transport these molecules to the borrelial surface is known. Accordingly, experiments supplemented by molecular modeling were undertaken to determine whether the orthologous LPT system identified in B. burgdorferi could transport lipoproteins and/or glycolipids to the borrelial outer membrane. Our combined observations strongly suggest that the LPT transport system does not transport lipoproteins to the surface. Molecular dynamic modeling, however, suggests that the borrelial LPT system could transport borrelial glycolipids to the outer membrane

The OspE-Related Proteins Inhibit Complement Deposition and Enhance Serum Resistance of Borrelia burgdorferi, the Lyme Disease Spirochete ▿

Borrelia burgdorferi, the Lyme disease spirochete, binds the host complement inhibitors factor H (FH) and FH-like protein 1 (FHL-1). Binding of FH/FHL-1 by the B. burgdorferi proteins CspA and the OspE-related proteins is thought to enhance resistance to serum-mediated killing. While previous reports have shown that CspA confers serum resistance in B. burgdorferi, it is unclear whether the OspE-related proteins are relevant in B. burgdorferi serum resistance when OspE is expressed on the borrelial surface. To assess the role of the OspE-related proteins, we overexpressed them in a serum-sensitive CspA mutant strain. OspE overexpression enhanced serum resistance of the CspA-deficient organisms. Furthermore, FH was more efficiently bound to the B. burgdorferi surface when OspE was overexpressed. Deposition of complement components C3 and C5b-9 (the membrane attack complex), however, was reduced on the surface of the OspE-overexpressing strain compared to that on the CspA mutant strain. These data demonstrate that OspE proteins expressed on the surface of B. burgdorferi bind FH and protect the organism from complement deposition and subsequent serum-mediated destruction

Borrelia burgdorferi complement regulator-acquiring surface protein 2 does not contribute to complement resistance or host infectivity.

Borrelia burgdorferi, the pathogen of Lyme disease, cycles in nature through Ixodes ticks and mammalian hosts. At least five Complement Regulator-Acquiring Surface Proteins (BbCRASPs) are produced by B. burgdorferi, which are thought to assist spirochetes in host immune evasion. Recent studies established that BbCRASP-2 is preferentially expressed in mammals, and elicits robust antibody response in infected hosts, including humans. We show that BbCRASP-2 is ubiquitously expressed in diverse murine tissues, but not in ticks, reinforcing a role of BbCRASP-2 in conferring B. burgdorferi defense against persistent host immune threats, such as complement. BbCRASP-2 immunization, however, fails to protect mice from B. burgdorferi infection and does not modify disease, as reflected by the development of arthritis. An infectious BbCRASP-2 mutant was generated, therefore, to examine the precise role of the gene product in spirochete infectivity. Similar to wild type B. burgdorferi, BbCRASP-2 mutants remain insensitive to complement-mediated killing in vitro, retain full murine infectivity and induce arthritis. Quantitative RT-PCR assessment indicates that survivability of BbCRASP-2-deficient B. burgdorferi is not due to altered expression of other BbCRASPs. Together, these results suggest that the function of a selectively expressed B. burgdorferi gene, BbCRASP-2, is not essential for complement resistance or infectivity in the murine host