Mitogenic Effect ofBartonella bacilliformison Human Vascular Endothelial Cells and Involvement of GroEL

Abstract

Bartonellae are bacterial pathogens for a wide variety of mammals. In humans, bartonellosis can result in angioproliferative lesions that are potentially life threatening to the patient, including bacillary angiomatosis, bacillary peliosis, and verruga peruana. The results of this study show that Bartonella bacilliformis, the agent of Oroya fever and verruga peruana, produces a proteinaceous mitogen for human vascular endothelial cells (HUVECs) that acts in a dose-dependent fashion in vitro with maximal activity at >72 h of exposure and results in a 6-to 20-fold increase in cell numbers relative to controls. The mitogen increases bromodeoxyuridine (BrdU) incorporation into HUVECs by almost twofold relative to controls. The mitogen is sensitive to heat and trypsin but is not affected by the lipopolysaccharide inhibitor polymyxin B. The mitogen does not affect caspase 3 activity in HUVECs undergoing serum starvation-induced apoptosis. The Bartonella mitogen was found in bacterial culture supernatants, the soluble cell lysate fraction, and, to a lesser degree, in insoluble cell fractions of the bacterium. In contrast, soluble cell lysate fractions from closely related B. henselae, although possessing significant mitogenicity for HUVECs, resulted in only about a twofold increase in cell numbers. Biochemical and immunological analyses identified GroEL as a participant in the observed HUVEC mitogenicity. A B. bacilliformis strain containing the intact groES-groEL operon on a multicopy plasmid was generated and used to demonstrate a correlation between HUVEC mitogenicity and GroEL levels in the lysate (r 2 ‫؍‬ 0.85). Antiserum to GroEL significantly inhibited mitogenicity of the lysate. Data also show that GroEL is located in the soluble and insoluble fractions (including inner and outer membranes) of the cell and is actively secreted by B. bacilliformis.Human infection by Bartonella species can result in angioproliferative lesions that stem from bacterium-induced hyperproliferation of the vasculature (1, 37). This pathology is unique among pathogenic bacteria and resembles Kaposi's sarcoma. The earliest study investigating this phenomenon identified a factor with a size of Ͼ12 kDa in the soluble fraction of a Bartonella bacilliformis cell homogenate that was specifically mitogenic for vascular endothelial cells (17). Human umbilical vein endothelial cells (HUVECs) treated with the factor showed proliferation rates approximately threefold greater than those in controls. The absence of cytotoxic effects coupled with heat sensitivity led the authors to conclude that the factor was not lipopolysaccharide (LPS). This study also demonstrated that the factor was angiogenic in a rat model. A subsequent study by this group showed that live B. bacilliformis could stimulate proliferation of HUVECs in cocultures (16).A study with Bartonella henselae showed enhanced proliferation and migration of HUVECs in coculture and identified a trypsin-sensitive proliferative factor detectable in the insoluble (cell wall) fraction of the bact...

Strategies of exploitation of mammalian reservoirs by Bartonella species

“…Far-Western blotting indicates that the Fc binding capacity was mediated by a protein of approximately 65kDa size, and N-terminal sequencing of this protein demonstrates its identity with the heat shock response protein GroEL. Western blotting of B. henselae cellular fractions indicates that GroEL was located in the cytoplasm and in the inner and outer membrane of the cell, as previously demonstrated for B. bacilliformis [59]. Expression of recombinant B. henselae GroEL conferred an Fc binding capacity on Escherichia coli (Figure 5).…”

Proteomic and Immunoblot Analyses of Bartonella quintana Total Membrane Proteins Identify Antigens Recognized by Sera from Infected Patients

“…Two of the proteins that we identified, GroEL and DnaK, are common heat shock proteins that also function as chaperones and thus are often membrane associated (7,37). Indeed, Bartonella bacilliformis has been shown to actively secrete GroEL (30). Other cytosolic proteins, including FusA, TypA, EF-Tu, and Tig, are ribosome-associated proteins that can be membrane associated during the biosynthesis of proteins destined for the periplasm or outer membrane (17).…”

“…The library was generated with a Sau3AI partial digest of B. quintana chromosomal DNA and the lambda-ZAP Express vector used according to the manufacturer's instructions (Stratagene, La Jolla, CA) and was screened by lifting plaques onto isopropyl-␤-D-thiogalactopyranoside (IPTG)-impregnated nitrocellulose (27), followed by immunoblotting, as previously described (30). The initial screening was performed for 3 h at 25°C using polyclonal rabbit anti-B.…”

Predominant Outer Membrane Antigens of Bartonella henselae

“…Nanospray-MS/MS identified this protein as OMP89, a possible surface protein containing a zinc metalloprotease domain (43). Protein 5 was identified as GroEL, a chaperonin that localizes to multiple subcellular fractions in Bartonella species (29). Amino acid sequence acquired for protein 6 did not match any protein sequence available in public databases.…”