Identification of Anaplasma marginale Type IV Secretion System Effector Proteins

Svetlana Lockwood,Paul A Beare,Robert A Heinzen,Shira L Broschat,Daniel E Voth,Kelly A Brayton,Wendy C Brown,Ulrike Gertrud Munderloh

doi:10.1371/journal.pone.0027724

Abstract

Background Anaplasma marginale, an obligate intracellular alphaproteobacterium in the order Rickettsiales, is a tick-borne pathogen and the leading cause of anaplasmosis in cattle worldwide. Complete genome sequencing of A. marginale revealed that it has a type IV secretion system (T4SS). The T4SS is one of seven known types of secretion systems utilized by bacteria, with the type III and IV secretion systems particularly prevalent among pathogenic Gram-negative bacteria. The T4SS is predicted to play an important role in the invasion and pathogenesis of A. marginale by translocating effector proteins across its membrane into eukaryotic target cells. However, T4SS effector proteins have not been identified and tested in the laboratory until now.ResultsBy combining computational methods with phylogenetic analysis and sequence identity searches, we identified a subset of potential T4SS effectors in A. marginale strain St. Maries and chose six for laboratory testing. Four (AM185, AM470, AM705 [AnkA], and AM1141) of these six proteins were translocated in a T4SS-dependent manner using Legionella pneumophila as a reporter system.ConclusionsThe algorithm employed to find T4SS effector proteins in A. marginale identified four such proteins that were verified by laboratory testing. L. pneumophila was shown to work as a model system for A. marginale and thus can be used as a screening tool for A. marginale effector proteins. The first T4SS effector proteins for A. marginale have been identified in this work.

Highlights

The type IV secretion system (T4SS) is found in a diverse set of microorganisms—including both Gram-negative and Gram-positive bacteria—that infect a variety of animal and plant hosts
Identification of potential effector proteins After comparing the properties of known T4SS effector proteins with the properties of A. marginale housekeeping genes (Tables S1, S2, S3, and S4), the following procedure was applied to select a subset of potential effector proteins
The resulting 21 proteins were ordered with higher ranking given to proteins with strong negative average hydropathy (Table 1)

Summary

Introduction

The type IV secretion system (T4SS) is found in a diverse set of microorganisms—including both Gram-negative and Gram-positive bacteria—that infect a variety of animal and plant hosts. Members of the order Rickettsiales comprise several animal and human pathogens. The identification of T4SSs naturally prompted a search for effector molecules secreted by these systems in order to identify mechanisms of virulence and pathogenesis. An obligate intracellular alphaproteobacterium in the order Rickettsiales, is a tick-borne pathogen and the leading cause of anaplasmosis in cattle worldwide. Complete genome sequencing of A. marginale revealed that it has a type IV secretion system (T4SS). The T4SS is one of seven known types of secretion systems utilized by bacteria, with the type III and IV secretion systems prevalent among pathogenic Gram-negative bacteria. The T4SS is predicted to play an important role in the invasion and pathogenesis of A. marginale by translocating effector proteins across its membrane into eukaryotic target cells. T4SS effector proteins have not been identified and tested in the laboratory until now

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