Proteomic And Bioinformatic Analysis Of The Bartonella Henselae Outer Membrane Proteins

Cat scratch disease (CSD) is a bacterial disease caused by Bartonella henselae which is an emerging zoonotic pathogen causing a wide range of disease manifestations in humans. Most people with CSD have been bitten or scratched by a cat and developed a mild infection at the point of injury. Lymph nodes, especially those around the head, neck, and upper limbs, become swollen. Additionally, a person with CSD may experience fever, headache, fatigue, and a poor appetite. Rare complications of B. henselae infection are bacillary angiomatosis, endocarditis and Parinaud's oculolandular syndrome.Despite there are significant advances in recent years, our understanding of Bartonella pathogenesis is still incomplete. Little is known about the disease pathogenesis and immunopathogenesis of Bartonella, diagnosis and immunoprophylaxis of human infection remains extremely challenging. To address this paucity of knowledge, we sought to identify potential membrane-associated virulence factors for adhesion, invasion and secretions of proteins, as well as protective and diagnostically relevant B. henselae antigens, by characterizing the outer membrane fraction and bioinformatic analysis of outer membrane proteome of B. henselae.We isolated the outer membrane proteins of B. henselae by using the ionic detergent lauryl sarcosine (SLS) and sodium carbonate, purification by two-dimensional electrophoresis with immobilized pH gradients (IPG Strips pH 3-10) in the first dimension, followed by protein identification using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Reference maps of OMPs were established and analyzed by software. It is possible to see that solubilization buffer with ASB-14 and ZWITTERGENT 3-10 can reveal significantly more spots especially in the base region of the 2-DE profiles, as shown by the increased number of spots with good resolution. Total 368 spots were calculated on the gel from the sarcosine-insoluble outer membrane fraction, and 94 distinct protein species from 176 spots were identified. In the outer membrane fraction by applying carbonate incubation, 471 spots were calculated on the gel and 259 were identified which represent 139 protein entries. There are 6 outer membrane proteins from the the sarcosine-insoluble outer membrane fraction comparing with 9 outer membrane proteins from samples of carbonate incubation. Among the total 10 outer membrane proteins variably expressed outer membrane protein porin, iron ion uptake associated proteins, surface antigens, putative drug efflux proteins, lipoprotein and peptidyl-prolyl cis-trans-isomerase. In an additional demonstration we show the value of bioinformatic analysis for 44 outer membrane proteins by prediction of domains and tertiary structures document the potential virulence factors comprising 5 iron ion adorption proteins, 5 adhesion proteins, 4 hemolysins, 6 aggruglobins, 7 autotransporter proteins, 2 drug efflux proteins, and 2 bacteria surface antigens.In summary, we established the 2-DE reference maps of the outer membrane subproteome of B. henselae by using the two different extraction methods which were complementary to each other in partly. Sodium carbonate extraction isolated low abundance and basic proteins better than the SLS extraction which preferred to enrich high abundance porins. Some efflux systems may present on B. henselae cells warranting further consideration as the potential ability to limit the access of antimicrobial agents to their targets. The identification and bioinformatic evaluation of these B. henselae outer membrane proteins should not only aid in the development of better diagnostic tests and better disease prevention but also provide insight into the pathogenesis of Bartonella.