ROUGH DRAFT authorea.com/64646

# Abstract

Francisella tularensis is a zoonotic bacterial pathogen that causes severe disease in a wide range of host animals, including humans. Well-developed mouse models of F. tularensis pathogenesis are available, but they do not meet the needs of all investigators. Instead, researchers are increasingly turning to insect model systems: (1) to allow for high-throughput that would be cost-prohibitive or ethically-questionable in mammals; (2) to enable studies of host-pathogen interactions in situations where mammalian facilities are unavailable; and (3) to provide valuable information about environmental persistence and transmission. However, the utility of previously-described insect hosts is limited because of temperature restriction, short lifespans, and concerns about the immunological status of insects mass-produced for other purposes. Here, we present a novel host species, the orange spotted (OS) cockroach (Blaptica dubia; Serville, 1839), that overcomes these limitations and is readily infected by F. tularensis. Intrahemocoel inoculation of OS cockroaches was accomplished using standard laboratory equipment and lethality was directly proportional to the number of bacteria injected. Disease progression differed in insects housed at low and high temperatures, indicating that the model could be useful for dissecting both virulence and transmission pathways. As in mammals, F. tularensis mutants lacking key components of the cell envelope or phagosomal escape pathway were attenuated in OS cockroaches. Finally, we examined the utility of this model in identifying antibiotics with in vivo activity against F. tularensis. Antibiotics were delivered by systemic injection or forced feeding; in the latter case, protection correlated with the oral bioavailability profile of each compound in mammals. Collectively, these results demonstrate that OS cockroaches are an important addition to investigators’ tool box that should facilitate discovery of factors that control F. tularensis virulence, immune evasion, and transmission while also providing a platform for early stage drug discovery and development.

# Importance

Invertebrate models of infection are critical to understanding host-pathogen interactions, having contributed countless discoveries to the biomedical enterprise. Despite the availability of multiple insect host species, we currently lack a robust, long-lived host that thrives at 37$$^\circ$$C, has low background rates of mortality, and can be easily reared and manipulated in the laboratory without specialized equipment. In the work presented here, we establish that the OS cockroach meets the above criteria and is a permissive experimental host for the model zoonotic pathogen Francisella tularensis. Further, relatively little is known regarding how F. tularensis survives and is transmitted by environmental arthropods. We show that F. tularensis virulence toward OS cockroaches varies according to temperature. Thus, investigators will be able to compare temperature-regulated virulence and transmission strategies within a single experimental host. Finally, we demonstrate that the OS cockroach can be used to test the protective efficacy and oral absorption of new therapeutic compounds. Our findings suggest that the OS cockroach will be an important experimental host species for F. tularensis and, perhaps, other bacterial pathogens.

# Introduction

Francisella tularensis is a Gram-negative bacterial pathogen capable of causing disease in a remarkably diverse array of hosts; at least 190 different species of mammals, 23 birds, 3 amphibians and 88 invertebrates are recognized as being susceptible to F. tularensis infection (Morner 2001). In addition, F. tularensis utilizes a wide variety of environmental arthropod vectors for transmission (Thelaus 2014, Lundström 2011, Asare 2010, Petersen 2009, Mani 2012, Goethert 2011, Broman 2011, Reese 2010, Reese 2011). In experimental animals, F. tularensis invades and replicates within both phagocytic and non-phagocytic cells (Santic 2013, Steele 2013, Chong 2010, Bradburne 2013) and several studies have demonstrated that F. tularensis survives engulfment by bacterivorous protists, often escaping from the food vacuole and replicating within the cytosol (Abd 2003, Lauriano 2004, El-Etr 2009). This ability to survive intracellularly is thought to contribute to the low infectious dose of F. tularensis, which is fewer than 10 bacteria for the highly-virulent strains (Ellis 2002). Due to this high infectivity and an accompanying high rate of mortality and morbidity, F. tularensis is of particular concern as an agent of biological terrorism and is therefore classified as a Tier 1 select agent by the US Centers for Disease Control (Dennis 2001). An attenuated live vaccine strain (LVS) originally was derived from a virulent isolate in the 1950s (Burke 1977). Desipte its name, LVS is not currently approved by the US Food and Drug Administration for standard human use because of safety and efficacy concerns. However, the LVS strain can be manipulated in biosafety level two laboratories (BSL2) and still causes rapid and severe disease in many hosts, allowing for F. tularensis pathogenesis studies without the need for BSL3 containment.