Abstract
Obligate intracellular bacteria have an arsenal of proteins that alter host cells to establish and maintain a hospitable environment for replication. Anaplasma phagocytophilum secrets Ankyrin A (AnkA), via a type IV secretion system, which translocates to the nucleus of its host cell, human neutrophils. A. phagocytophilum-infected neutrophils have dramatically altered phenotypes in part explained by AnkA-induced transcriptional alterations. However, it is unlikely that AnkA is the sole effector to account for infection-induced transcriptional changes. We developed a simple method combining bioinformatics and iTRAQ protein profiling to identify potential bacterial-derived nuclear-translocated proteins that could impact transcriptional programming in host cells. This approach identified 50 A. phagocytophilum candidate genes or proteins. The encoding genes were cloned to create GFP fusion protein-expressing clones that were transfected into HEK-293T cells. We confirmed nuclear translocation of six proteins: APH_0062, RplE, Hup, APH_0382, APH_0385, and APH_0455. Of the six, APH_0455 was identified as a type IV secretion substrate and is now under investigation as a potential nucleomodulin. Additionally, application of this approach to other intracellular bacteria such as Mycobacterium tuberculosis, Chlamydia trachomatis and other intracellular bacteria identified multiple candidate genes to be investigated.
Highlights
Anaplasma phagocytophilum is an obligate intracellular bacterium of human neutrophils
Since these events occur in the nucleus, we developed an unbiased computational approach to identify potential nucleomodulins encoded in intracellular bacterial genomes based on their likelihood for translocation into the host cell nucleus and applied this to the A. phagocytophilum HZ strain genome (Supplemental Figure 1)
PHAGOCYTOPHILUM PROTEINS TARGETED TO THE HOST CELL NUCLEUS Of 1264 proteins and hypothetical proteins examined by the bioinformatics algorithm, 123 were identified by ProtComp as nuclear-localized; 3 of these were classified in PSORTb as potentially nuclear membrane-associated; after analysis of NLSdb and screening for NLSm and NLSb, 7 candidate proteins had a total Nuclear score >1 (Table 1)
Summary
Anaplasma phagocytophilum is an obligate intracellular bacterium of human neutrophils. The neutrophil is an unlikely host as it creates an intracellular milieu that is a highly inhospitable environment for bacterial survival. A. phagocytophilum requires the neutrophil for propagation and survives by altering the cellular antimicrobial properties while paradoxically increasing proinflammatory functions (Banerjee et al, 2000; Carlyon et al, 2002; Borjesson et al, 2005; Choi et al, 2005; Carlyon and Fikrig, 2006). There is increasing evidence to suggest that the bacterium accomplishes this with coordinated reprogramming of neutrophil gene transcription by reorganizing large regions of host cell chromatin (Sinclair et al, 2014). Our laboratory investigated the effect of infection on the transcriptional repression of CYBB, encoding gp91phox (Garcia-Garcia et al, 2009a,b)
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