Date Published: September 27, 2018
Publisher: Public Library of Science
Author(s): Emily M. Carpinone, Zhiru Li, Michael K. Mills, Clemence Foltz, Emma R. Brannon, Clotilde K. S. Carlow, Vincent J. Starai, Zhao-Qing Luo.
Wolbachia is an unculturable, intracellular bacterium that persists within an extremely broad range of arthropod and parasitic nematode hosts, where it is transmitted maternally to offspring via vertical transmission. In the filarial nematode Brugia malayi, a causative agent of human lymphatic filariasis, Wolbachia is an endosymbiont, and its presence is essential for proper nematode development, survival, and pathogenesis. While the elucidation of Wolbachia:nematode interactions that promote the bacterium’s intracellular persistence is of great importance, research has been hampered due to the fact that Wolbachia cannot be cultured in the absence of host cells. The Wolbachia endosymbiont of B. malayi (wBm) has an active Type IV secretion system (T4SS). Here, we have screened 47 putative T4SS effector proteins of wBm for their ability to modulate growth or the cell biology of a typical eukaryotic cell, Saccharomyces cerevisiae. Five candidates strongly inhibited yeast growth upon expression, and 6 additional proteins showed toxicity in the presence of zinc and caffeine. Studies on the uptake of an endocytic vacuole-specific fluorescent marker, FM4-64, identified 4 proteins (wBm0076 wBm00114, wBm0447 and wBm0152) involved in vacuole membrane dynamics. The WAS(p)-family protein, wBm0076, was found to colocalize with yeast cortical actin patches and disrupted actin cytoskeleton dynamics upon expression. Deletion of the Arp2/3-activating protein, Abp1p, provided resistance to wBm0076 expression, suggesting a role for wBm0076 in regulating eukaryotic actin dynamics and cortical actin patch formation. Furthermore, wBm0152 was found to strongly disrupt endosome:vacuole cargo trafficking in yeast. This study provides molecular insight into the potential role of the T4SS in the Wolbachia endosymbiont:nematode relationship.
After leprosy, lymphatic filariasis is the leading cause of permanent disability, afflicting at least 150 million people worldwide [1, 2]. The World Health Organization reports that 1.23 billion people in 58 countries are at risk for developing the disease . Lymphatic filariasis results from the mosquito-borne transmission of three distinct pathogenic nematodes: Wuchereria bancrofti, Brugia timori and Brugia malayi. In the early stages of the infection, anti-parasitic agents such as diethylcarbamazine and ivermectin have proved to be effective in eliminating immature worms and have been used in mass treatment programs . Unfortunately, adult worms which are largely responsible for the pathology associated with infection cannot be easily treated by early-stage anti-parasitic drugs [4, 5]. With the discovery that these filarial nematodes require the presence of an obligate intracellular, Gram-negative bacterial endosymbiont of the Wolbachia genus to survive and reproduce, effective clearance of filarial infections has been achieved with antibiotic treatment [4–8]. Accordingly, significant efforts to understand the interactions of Wolbachia with nematodes at the molecular level have been undertaken to identify additional potential drug targets .
Though some progress has been made in understanding the Wolbachia:host relationship at the molecular level, much remains unknown about the mechanisms by which Wolbachia persists in its nematode or insect hosts. The use of S. cerevisiae as a highly tractable surrogate eukaryotic host provides an avenue to rapidly screen wBm putative T4SS effectors that may manipulate eukaryotic cell physiology and membrane trafficking pathways likely required for the persistence of Wolbachia in host cells. Historically, the S. cerevisiae model has successfully identified virulence factors from human pathogens that, upon expression in yeast, induce a growth defect and alter eukaryotic physiology, as is the case with the identification of secreted proteins from a variety of pathogenic bacteria, including Chlamydia trachomatis, Legionella pneumophila, Pseudomonas aeruginosa, and Salmonella enterica [27, 29, 77, 78]. Recently, S. cerevisiae was used to identify fourteen candidate effectors of the Wolbachia endosymbiont of Drosophila melanogaster, wMel, with each candidate effector inducing growth defects in S. cerevisiae . Collectively, these data have verified that S. cerevisiae is a valuable tool to evaluate the molecular function of a number of Wolbachia-secreted proteins. Therefore, we have employed S. cerevisiae to screen 47 predicted wBm effectors to identify those that manipulate eukaryotic physiology. Due to the ability of some of these proteins to induce growth defects and alter the cellular processes in S. cerevisiae (Table 2), we propose that they may function as secreted effectors of wBm and may be necessary for germline cell invasion and persistence of wBm in its nematode host. Strikingly, two of these proteins–wBm0076 and wBm0152—are particularly strong modulators of yeast physiology.