Ehrlichia chaffeensis replication sites in adult Drosophila melanogaster

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Abstract

Ehrlichia chaffeensis is a Gram-negative, obligate intracellular bacterium which causes the tick-borne disease human monocytic ehrlichiosis. In vertebrates, E. chaffeensis replicates in monocytes and macrophages. However, no clear cell or tissue tropism has been defined in arthropods. Our group identified two host genes that control E. chaffeensis replication and infection in vivo in Drosophila, Uridine cytidine kinase and separation anxiety. Using the UAS-GAL4 RNAi system, we generated F1 flies (UAS-gene of interestRNAi x tissue-GAL4 flies) that have Uck2 or san silenced in ubiquitous or tissue-specific fashion. When Uck2 or san were suppressed in the hemocytes or in the fat body, E. chaffeensis replicated poorly and caused significantly less severe infections. Silencing of these genes in the eyes, wings, or the salivary glands did not impact fly susceptibility or bacterial replication. Our data suggest that in Drosophila, E. chaffeensis replicates within the hemocytes, the insect homolog of mammalian macrophages, and in the fat body, the liver homolog of mammals.

Introduction

Human monocytic ehrlichiosis (HME) is a tick-borne, zoonotic disease caused by the Gram-negative, obligate intracellular bacterium, Ehrlichia chaffeensis. E. chaffeensis is primarily vectored by Amblyomma americanum (lone star tick) (Anderson et al., 1993) and is transmitted transstadially in ticks (Parola et al., 2005). A. americanum has a 3-host life cycle. For progression from one stage to the next (larva to nymph to adult), the tick requires a vertebrate blood meal (Parola et al., 2005). The major natural reservoir of E. chaffeensis in the United States is white-tailed deer (Odocoileus virginianus) (Dawson et al., 1994, Lockhart et al., 1997). However, several other vertebrates, too, act as reservoirs including domestic dog (Yu et al., 2008), domestic goat (Dugan et al., 2000), white-footed mouse (Magnarelli et al., 1997), red fox (Davidson et al., 1999), raccoon (Dugan et al., 2005), and coyote (Kocan et al., 2000). Humans can also become accidental hosts when bitten by ticks. In humans, the bacteria are monocytotropic, meaning they are primarily found in monocytes and macrophages, and there is a good understanding of where the bacteria replicate (Sotomayor et al., 2001). Although it is clear that ticks can transmit Ehrlichia organisms to vertebrate hosts (Ewing et al., 1995, Varela-Stokes, 2007), and Ehrlichia bacteria have been detected in the salivary glands microscopically (Smith et al., 1976) and by PCR (Karim et al., 2012), it is less clear about bacteria replication in ticks. Although genetic tools for working with ticks are currently being developed (Pagel Van Zee et al., 2007), the available tools do not begin to approach those that are available in other arthropods such as Drosophila melanogaster.

In adult D. melanogaster, hemocytes contribute to host immune defenses against E. chaffeensis (Luce-Fedrow et al., 2009). In vertebrates, monocytes and macrophages contribute to host resistance (Chapes and Ganta, 2008, Ganta et al., 2002) even though the bacteria are monocytotropic. Our group has discovered that D. melanogaster genes, separation anxiety (san) and Uridine cytidine kinase 2 (Uck2), are required for E. chaffeensis infections in flies (Von Ohlen et al., 2012). Flies carrying mutations in the coding regions of these genes do not support infection after needle injection (Von Ohlen et al., 2012). Therefore, to see if bacterial replication in arthropods parallels bacterial replication in vertebrates after needle injection, we took advantage of the Drosophila UAS-GAL4 RNAi system (Dietzl et al., 2007) that allowed for tissue-specific silencing of san and Uck2 to determine host sites of bacterial replication. Here, we show that similar to vertebrate hosts, in Drosophila the bacteria require optimal host conditions (i.e. expression of Uck2 or san) in the immune tissues, hemocytes, and fat body, for optimal replication.

Section snippets

Cell lines and E. chaffeensis infections

E. chaffeensis (Arkansas isolate) was propagated in DH82 cells (American Type Culture collection, #CRL-10389, Rockville, Md.). The DH82 cells were grown in Eagle's minimal essential medium supplemented with 3.5% fetal bovine serum (Atlanta Biologicals, Atlanta, GA), 3.5% NuSerum (BD, Franklin Lakes, NJ), and Glutamine plus (2 mM, Atlanta Biologicals) (EMEM7). Cells were grown at 37 °C in an 8% CO2/92% air atmosphere. The level of infection was determined by examining cyto-centrifuged cells

Uck2 and san are required for in vivo E. chaffeensis replication in adult D. melanogaster

E. chaffeensis is capable of infecting and completing its life cycle in Drosophila S2 cells and adult flies (Luce-Fedrow et al., 2008, Luce-Fedrow et al., 2009). Recent work by our group found that several host genes control the replication of Ehrlichia in vivo (Von Ohlen et al., 2012). We found that flies carrying mutations in coding regions of these genes were poorly infected by E. chaffeensis. In particular, 2 genes that are relevant to humans and ticks were chosen for this study, Uck2 and

Discussion

E. chaffeensis is transmitted from ticks to vertebrate hosts when the tick takes a blood meal (Paddock and Childs, 2003). One remaining question is where do the bacteria replicate in the tick? Because the genetic tools in ticks have not been developed to the same extent as they have in Drosophila, we addressed a more general question about where the bacteria replicate in dipteran arthropods. Indeed, this study provides insights about E. chaffeensis replication in arthropods. Our data confirm

Acknowledgements

We would like to thank Ms. Rachel Nichols, Ms. Victoria Davidson, and Mr. Taylor Kinney for their help with the flies. We thank Drs. Alison Luce-Fedrow and Teresa Ortega for valuable help in the lab. We thank Dr. Roman Ganta for his valuable discussions about E. chaffeensis. This project has been supported in part by NIH grants AI088070, AI55052, AI052206, RR16475, GM103418 and RR017686, the Kansas Agriculture Experiment Station Animal Health Project grant 481848, the Kansas Space Grant

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