Interaction Of Cytoskeletal Proteins With Infection Of Bartonella Henselae Into Epithelial Cells

Bartonella henselae (B. henselae) is a small, fastidious, intracellularGram-negative bacteria that parasites in the erythrocyte, vascular endothelial cells,epithelial cells and phagocytic cells. Erythrocyte parasitism is the hallmark ofBartonella infection. B. henselae is the most common zoonotic pathogen worldwidewith wide disease spectrum. Cats are the major reservoir host of B. henselae. Humaninfections of B. henselae are mainly transmitted from cats by the cat fea or directly bycat scratch or bite, resulting in cat-scratch disease (CSD), which mainly goes with thelocal lymph nodes swollen and skin injury. For immunocompromised infectedpatients, B. henselae will cause bacillary angiomatosis (BA), endocarditis,neuroretinitis and bacillary peliosis (BP). However, bartonellosis has still beendefined as a neglected disease. In China, there are a large number of misdiagnosedcases. Researches of bartonellosis in China, regarding to the diagnostic techniques,epidemiology and pathogenesis, are waiting to be studied. Hitherto, the host bindingfactors for B. henselae infection and its association with pathogeneisis needs to befurther studied.The aim of the present study is to explore the mechanism of B. henselaeinfection into epithelial cells, following researches were performed:1. Staining of B. henselae with carboxyfluorescein diacetatesuccinimidyl ester (CFDA-SE) for tracking infection in erythrocytesand epithelial cellsIn this study, B. henselae was labeled with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) to track the infection in erythrocytes and epithelialcells. Result of flow cytometry confirmed that stainning of10min with CFDA-SE caneffectively label bacillus. Stained B. henselae was confirmed to be able to penetrateinto erythrocytes. Flow cytometry assay identifed4.86%,3.85%and8.47%of theerythrocytes were infected with stained bacilli under multiplicity of infection (MOI)from1to100per erythrocyte, respectively. For HeLa cell infectioin, the invasomestructure and the single bacterial residing in perinuclear could be observed. Paralleltests using untreated bacteria confirmed that CFDA-SE staining did not result in sideeffects on the infectivity of B. henselae. This technique offers an easy way ofidentifying the intracellular localization of bacteria in erythrocytes and epithelial cellswithout the use of antibodies. The efficient invasion of CFDA-SE staining B. henselaein erythrocytes and epithelial cells can be followed up on a time-to-time basis toobtain information on the localization of B. henselae in early infection.2. The effects of cytoskeleton proteins on invasion of B. henselae inHeLa cell(1) Identification of binding candidates with B. henselae invasion inHeLa cellIn this study, the out membrane protein (OMP) of B. henselae were labeled withbiotin. Binding proteins in HeLa cells were screened by using Pull-Down strategybased on the biotin-avidin strategy. Results showed several membrane proteins ofHeLa cells, molecular weight ranged from44to70-KD, bind with B. henselae. Theprotein bands were cut from the separating gel and analyzed by the tandem massspectrometry. Peptides were identifed by searching the HUMAN.v3.87.fasta databasewith the Bioworks Browser3.3software. The identified proteins are manilycytoskeleton-associated protein (keratin14, keratin6and F-actin). Therefore, theintermediate filament and microfilament proteins were selected in our further study toinvestigate the association of cytoskeleton and the intracellular infection of B.henselae. (2) Regulation of genes transcription and arrangement of cytoskeletalprotein by B. henselae infectionIn this study, relative quantitiative PCR and western blot were used to investigateexpression changes of the corresponding protein genes induced by B. henselaeinfection. Meanwhile, the morphological changes of the cytoskeleton in the infectedHeLa cells were detected by fluorescent microscope and whole mount cell electronmicroscope. Results showed that the infection of B. henselae up-regulated thecytoskeletal protein gene transcription, and cytokeratin KRT6elevated mostsignificantly. Fluorescence staining showed that microfilaments are rearranged andtwined around the bacterial aggregation. But the intermediate filaments aredepolymerized by B. henselae infection. It was speculated that the up-regulation ofcytoskeleton proteins, especially cytokeratins, is the response of anti-infection of hostcell, which might play the role to restrict the further spread of intracelluar bacillus.(3) Role of intermediate filament and microfilament on the infectionof B. henselae in HeLa cellsIn order to characterise the cytoskeletal components involved in the infection ofB. henselae, drugs, known to caused depolymerisation of intermediate filament(EGTA) and microfilament (cytochalasin B) or stabilization of microfilament(Phalloidin), were used. The results showed that treatment with EGTA promotes theintracellular infection of B. henselae. However, F-actin depolymerising agent-cytochalasin B resultes in a concentration-dependent inhibition of B. henselaeinfection into the epithelial cells. Similarly, treatment with Phalloidin significantdecreases the invasion of B. henselae. This study indicated that B. henselae infectionis F-actin-dependent. However, cytoskeletal intermediate filament playes an adverserole on B. henselae infection.